U.S. patent application number 11/471343 was filed with the patent office on 2007-03-08 for synergistic polyphenol compounds, compositions thereof, and uses thereof.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. Invention is credited to I. Bernard Weinstein.
Application Number | 20070054868 11/471343 |
Document ID | / |
Family ID | 37830742 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070054868 |
Kind Code |
A1 |
Weinstein; I. Bernard |
March 8, 2007 |
Synergistic polyphenol compounds, compositions thereof, and uses
thereof
Abstract
The present invention relates to Synergistic Polyphenol
Compounds, compositions thereof, and methods for treating or
preventing cancer in a subject, the methods comprising
administering to a subject an effective amount of a Synergistic
Polyphenol Compound or composition thereof.
Inventors: |
Weinstein; I. Bernard; (New
York, NY) |
Correspondence
Address: |
WilmerHale/Columbia University
399 PARK AVENUE
NEW YORK
NY
10022
US
|
Assignee: |
The Trustees of Columbia University
in the City of New York
New York
NY
|
Family ID: |
37830742 |
Appl. No.: |
11/471343 |
Filed: |
June 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60692108 |
Jun 20, 2005 |
|
|
|
60787305 |
Mar 29, 2006 |
|
|
|
Current U.S.
Class: |
514/27 ; 514/449;
514/456; 514/49 |
Current CPC
Class: |
A61K 31/7048 20130101;
A61K 31/7048 20130101; A61K 31/353 20130101; A61K 31/337 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 31/337 20130101; A61K 45/06 20130101; A61K 31/353
20130101; A61K 31/7072 20130101; A61K 31/7072 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/027 ;
514/049; 514/449; 514/456 |
International
Class: |
A61K 31/7072 20070101
A61K031/7072; A61K 31/7048 20070101 A61K031/7048; A61K 31/353
20070101 A61K031/353; A61K 31/337 20070101 A61K031/337 |
Claims
1. A composition comprising two or more compounds or
pharmaceutically acceptable salts thereof, wherein the compounds
are selected from a catechin, including but not limited to
(-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin
gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate; a
phenolic acid, including but not limited to gallic acid, caffeic
acid and ellagic acid; a bioflavanoid, including but not limited to
an anthocyanin, apigenin, and quercetin; and a complex polyphenol,
including but not limited to, a tannin and a lignan, and any
combination thereof, and wherein the amounts of the two or more
compounds are together synergistically effective to treat or
prevent colorectal cancer or liver cancer.
2. The composition of claim 1, wherein the compounds are selected
from (+)-polyphenol, (-)-epicatechin, (-)-epicatechin gallate,
(-)-gallocatechin gallate, (-)-epigallocatechin and
(-)-epigallocatechin gallate.
3. The composition of claim 1 comprising (-)-epigallocatechin
gallate.
4. The composition of claim 2 further comprising
(-)-epigallocatechin.
5. The composition of claim 2 further comprising (-)-epicatechin
gallate.
6. The composition of claim 2 further comprising (-)-gallocatechin
gallate.
7. The composition of claim 2 further comprising
(-)-epicatechin.
8. The composition of claim 2 further comprising
(+)-polyphenol.
9. The composition of claim 1, wherein the two or more compounds
are together synergistically effective to treat or prevent
colorectal cancer.
10. The composition of claim 1, wherein the two or more compounds
are together synergistically effective to treat or prevent liver
cancer.
11. The composition of claim 2 consisting essentially of
(-)-epigallocatechin gallate and (-)-epigallocatechin.
12. The composition of claim 2 consisting essentially of
(-)-epigallocatechin gallate and (-)-epicatechin gallate.
13. The composition of claim 2 consisting essentially of
(-)-epigallocatechin gallate and (-)-gallocatechin gallate.
14. The composition of claim 2 consisting essentially of
(-)-epigallocatechin gallate and (-)-epicatechin.
15. The composition of claim 2 consisting essentially of
(-)-epigallocatechin gallate and (+)-polyphenol.
16. The composition of claim 1 or 2 wherein one or more of the
compounds are in isolated form.
17. The composition of claim 14, wherein the ratio of
(-)-epicatechin to (-)-epigallocatechin gallate to is from about 5
to about 2.
18. The composition of claim 14, wherein the ratio of
(-)-epicatechin to (-)-epigallocatechin gallate to is from about 5
to about 1.
19. A method for treating or preventing colorectal cancer or liver
cancer in a subject, the method comprising administering to the
subject an effective amount of the composition of claim 1.
20. The method of claim 19, wherein the composition of claim 1
comprises (-)-epigallocatechin gallate.
21. The method of claim 19, wherein the composition further
comprises (-)-epicatechin
22. A method for treating colorectal cancer or liver cancer in a
subject, the method comprising: (a) administering to the subject a
first compound or pharmaceutically acceptable salt thereof,
selected from a catechin, including but not limited to
(-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin
gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate; a
phenolic acid, including but not limited to gallic acid, caffeic
acid and ellagic acid; a bioflavanoid, including but not limited to
an anthocyanin, apigenin, and quercetin; and a complex polyphenol,
including but not limited to, a tannin and a lignan, and any
combination thereof; and (b) administering to the subject a second
compound or pharmaceutically acceptable salt thereof, selected from
a catechin, including but not limited to (-)-epicatechin,
(-)-epicatechin gallate, (-)-gallocatechin gallate,
(-)-epigallocatechin and (-)-epigallocatechin gallate; a phenolic
acid, including but not limited to gallic acid, caffeic acid and
ellagic acid; a bioflavanoid, including but not limited to an
anthocyanin, apigenin, and quercetin; and a complex polyphenol,
including but not limited to, a tannin and a lignan, and any
combination thereof, wherein the first and second compounds are
different and wherein the amounts of the first and second compounds
administered are together synergistically effective to treat or
prevent colorectal cancer or liver cancer.
23. The method of claim 22, wherein the first compound is
(-)-epigallocatechin gallate.
24. The method of claim 23, wherein the second compound is
(-)-epicatechin.
25. The method of claim 24, wherein the ratio of the amount of
(-)-epicatechin administered to the amount of (-)-epigallocatechin
gallate administered to is about 5 to about 2.
26. The method of claim 23, wherein the ratio of the amount of
(-)-epicatechin administered to the amount of (-)-epigallocatechin
gallate administered to is about 5 to about 1.
27. The method of claim 22 wherein the first compound is
administered prior to the second compound.
28. The method of claim 22 wherein the first compound is
administered at some time after the second compound.
29. The method of claim 22 wherein the first compound and second
compound are administered concurrently.
30. The method of claim 22 wherein at least one of the first
compound and the second compound are in isolated form.
31. The method of claim 22, further comprising the administration
of another anticancer agent.
32. The method of claim 31, wherein the other anticancer agent is
taxol or 5-fluorouracil.
Description
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 60/692,108,
filed Jun. 20, 2005, and U.S. Provisional Patent Application No.
60/787,305, filed Mar. 29, 2006, each of which are hereby
incorporated by reference herein in their entirety.
[0002] All patents, patent applications and publications cited
herein are hereby incorporated by reference in their entirety. The
disclosures of these publications in their entireties are hereby
incorporated by reference into this application in order to more
fully describe the state of the art as known to one skilled therein
as of the date of the invention described and claimed herein.
[0003] This patent disclosure contains material that is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or the
patent disclosure as it appears in the U.S. Patent and Trademark
Office patent file or records, but otherwise reserves any and all
copyright rights.
1. FIELD OF THE INVENTION
[0004] The present invention relates to Synergistic Polyphenol
Compounds, compositions thereof, and methods for treating or
preventing cancer in a subject, the methods comprising
administering to a subject an effective amount of a Synergistic
Polyphenol Compound or composition thereof.
2. BACKGROUND OF THE INVENTION
[0005] Cancer is second only to cardiovascular disease as a cause
of death in the United States. The American Cancer Society
estimated that in 2002, there were 1.3 million new cases of cancer
and 555,000 cancer-related deaths. There are currently over 10
million living Americans who have been diagnosed with cancer and
the NIH estimates the direct medical costs of cancer as over $100
billion per year with an additional $100 billion in indirect costs
due to lost productivity--the largest such costs of any major
disease.
[0006] Modalities useful in the treatment of cancer include
chemotherapy, radiation therapy, surgery and biological therapy (a
broad category that includes gene-, protein- or cell-based
treatments and immunotherapy).
[0007] Despite the availability to the clinician of a variety of
anticancer agents, traditional chemotherapy has many drawbacks.
Almost all anticancer agents are toxic, and chemotherapy can cause
significant, and often dangerous, side effects, including severe
nausea, bone marrow depression, liver, heart and kidney damage, and
immunosuppression. Additionally, many tumor cells eventually
develop multi-drug resistance after being exposed to one or more
anticancer agents. As such, single-agent chemotherapy is curative
in only a very limited number of cancers. Most chemotherapeutic
drugs act as anti-proliferative agents, acting at different stages
of the cell cycle. Since it is difficult to predict the pattern of
sensitivity of a neoplastic cell population, or the current stage
of the cell cycle that a cell happens to be in, it is common to use
multi-drug regimens in the treatment of cancer, which are typically
more effective, but also more toxic than single-drug chemotherapy
regimens.
[0008] The EGFR subfamily of RTKs includes, in addition to EGFR and
HER2, the receptors HER3 (erbB3) and HER4 (erbB4). Binding of
specific ligands to EGFR, HER3, and HER4 results in receptor homo-
and heterodimerization thus activating the tyrosine kinase
activities of these receptors, including HER2 which lacks its own
ligand binding domain. This leads to activation of downstream
signaling pathways including the MAPK and PI3K pathways, and the
expression of genes that enhance cell proliferation. Aberrant
expression and constitutive activation of members of the EGFR
family have been observed in several types of human malignancies,
including colorectal cancer. Thus, human colorectal carcinoma often
displays overexpression of both EGFR and HER2. Increased levels of
expression of HER3 mRNA and protein are also frequently seen in
colorectal carcinoma. Although HER3 does not itself possess an
active kinase, the HER2/HER3 heterodimer can play a critical role
in enhancing the growth of cancer cells.
[0009] It is known that the HER2/HER3 pathway in colon cancer cells
induces the expression of COX-2 mRNA and protein, and results in
the accumulation of PGE.sub.2, a major metabolic product of COX-2.
COX-2 expression is also induced by several oncogenes, tumor
promoters, cytokines, and growth factors. In addition, COX-2
expression increases during colon carcinogenesis and it is
frequently overexpressed in human colon carcinoma. Although the
precise mechanism by which increased expression of COX-2 enhances
colon carcinogenesis is not known, it is possible that the
production of PGE.sub.2 by this enzyme plays an important role,
since it is known that treatment of colon cancer cells with
PGE.sub.2 results in transactivation of the EGFR, activation of ERK
and stimulation of cell proliferation.
[0010] Previous studies indicate that the naturally occurring
polyphenol EGCG can inhibit activation of the tyrosine kinase
activities of several RTKs including EGFR, HER2, PDGFR, and FGFR.
In addition to acting on cell surface receptors, EGCG can also
directly target intracellular signaling molecules. Thus, EGCG can
directly inhibit the subcellular kinase activities of ERK and Akt
in extracts of immortalized cervical cells.
[0011] There is increasing evidence for a complex positive feedback
circuitry between the EGFR signaling system, COX-2, and PGE.sub.2,
which can stimulate the growth of colon cancer cells. Thus,
activation of the EGFR and related RTKs leads to the induction of
COX-2 expression and the synthesis of PGE.sub.2. Treatment of colon
cancer cells with PGE.sub.2, in turn, leads to rapid
phosphorylation of the EGFR and ERK activation, and stimulation of
cell growth. On the other hand, inactivation of EGFR kinase with
selective inhibitors causes a decrease in PGE.sub.2-induced ERK
activation and cell proliferation. However, the precise mechanisms
by which PGE.sub.2 leads to activation of the EGFR and the RAS/MAPK
pathway are not currently known.
[0012] Amongst the members of the EGF receptor family, HER3 is the
most efficient activator of PI3K since it contains six docking
sites for the p85 protein, an adaptor subunit of PI3K. It has been
reported that in colon cancer cells PGE.sub.2 led to an increase in
cell proliferation and motility via activation of the PI3K/Akt
pathway. In addition, activation of HER3 by heregulin strongly
induces COX-2 expression and PGE.sub.2 synthesis in colon cancer
cells. Further, it has also been reported that oral infusion of a
green tea mixture inhibits the development and progression of
prostate cancer in the TRAMP mouse model of this disease. This was
associated with a reduction in the level of IGF-1 and an increase
in IGFBP-3 in both the serum and prostate tissue of the treated
mice and these results indicate that a non-toxic dose of green tea
polyphenols can affect the IGF/IGF-R system in vivo.
[0013] Accordingly, there exists a need for inhibitors of the
enzymatic activity of COX-2 for the prevention and treatment of
colon cancer and other types of cancer, wherein such inhibitors
have improved therapeutic indices. This invention addresses that
need.
[0014] The recitation of any reference in this application is not
an admission that the reference is prior art to this
application.
3. SUMMARY OF THE INVENTION
[0015] In one aspect, the invention provides Synergistic Polyphenol
Compositions comprising two or more compounds or pharmaceutically
acceptable salts thereof, wherein the compounds are selected from:
a catechin, including but not limited to (-)-epicatechin,
(-)-epicatechin gallate, (-)-gallocatechin gallate,
(-)-epigallocatechin and (-)-epigallocatechin gallate; a phenolic
acid, including but not limited to gallic acid, caffeic acid and
ellagic acid; a bioflavanoid, including but not limited to an
anthocyanin, apigenin, and quercetin; and a complex polyphenol,
including but not limited to, a tannin and a lignan, and any
combination thereof, wherein the amounts of the two or more
compounds are together synergistically effective to treat
cancer.
[0016] In another aspect, the invention provides Synergistic
Polyphenol Compositions consisting essentially of two or more
compounds or pharmaceutically acceptable salts thereof, wherein the
compounds are selected from: a catechin, including but not limited
to (-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin
gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate; a
phenolic acid, including but not limited to gallic acid, caffeic
acid and ellagic acid; a bioflavanoid, including but not limited to
an anthocyanin, apigenin, and quercetin; and a complex polyphenol,
including but not limited to, a tannin and a lignan, and any
combination thereof, and wherein the amounts of the two or more
compounds are together synergistically effective to treat
colorectal cancer or liver cancer.
[0017] In a further aspect, the invention provides methods for
treating or preventing cancer in a subject, the method comprising
administering to the subject an effective amount of a Synergistic
Polyphenol Composition.
[0018] In still another aspect, the invention provides methods for
treating or preventing cancer in a subject, the method comprising:
(a) administering to the subject a first compound or
pharmaceutically acceptable salt thereof, wherein the first
compound is selected from: a catechin, including but not limited to
(-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin
gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate; a
phenolic acid, including but not limited to gallic acid, caffeic
acid and ellagic acid; a bioflavanoid, including but not limited to
an anthocyanin, apigenin, and quercetin; and a complex polyphenol,
including but not limited to, a tannin and a lignan, and any
combination thereof, and (b) administering to the subject a second
compound or pharmaceutically acceptable salt thereof, wherein the
second compound is selected from: a catechin, including but not
limited to (-)-epicatechin, (-)-epicatechin gallate,
(-)-gallocatechin gallate, (-)-epigallocatechin and
(-)-epigallocatechin gallate; a phenolic acid, including but not
limited to gallic acid, caffeic acid and ellagic acid; a
bioflavanoid, including but not limited to an anthocyanin,
apigenin, and quercetin; and a complex polyphenol, including but
not limited to, a tannin and a lignan, and any combination thereof,
wherein the first and second compounds are different and wherein
the amounts of the first and second compounds administered are
together synergistically effective to treat or prevent cancer.
[0019] The invention provides methods for treating or preventing
cancer in a subject which comprises administering to a subject an
effective amount of a Synergistic Polyphenol Composition or one for
more Synergistic Polyphenol Compounds.
[0020] The present invention may be understood more fully by
reference to the following detailed description and illustrative
examples, which are intended to exemplify non-limiting embodiments
of the invention.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the results of cell viability assays which were
used to determine the growth inhibitory effects of a combination of
(-)-epigallocatechin gallate and (-)-epicatechin on HT29 human
colon cancer cells. The x-axis of FIG. 1A represents the
(-)-epigallocatechin gallate concentration, while the y-axis
represents cell viability as a percentage. The lines on the graph
in FIG. 1A from top to bottom, represent the following:
(-)-epicatechin alone, (-)-epicatechin+0.1 .mu.g/mL
(-)-epigallocatechin gallate, (-)-epicatechin+1.0 .mu.g/mL
(-)-epigallocatechin gallate, (-)-epicatechin+10 .mu.g/mL
(-)-epigallocatechin gallate, and (-)-epicatechin+20 .mu.g/mL
(-)-epigallocatechin gallate. The x-axis of FIG. 1B represents the
(-)-epicatechin concentration, while the y-axis represents cell
viability as a percentage. The lines on the graph in FIG. 1B from
top to bottom, represent the following: (-)-epigallocatechin
gallate alone, (-)-epigallocatechin gallate+1.0 .mu.g/mL
(-)-epicatechin, (-)-epigallocatechin gallate+10 .mu.g/mL
(-)-epicatechin, (-)-epigallocatechin gallate+50 .mu.g/mL
(-)-epicatechin, (-)-epigallocatechin gallate+100 .mu.g/mL
(-)-epicatechin.
[0022] FIG. 2 shows the effects of (-)-epigallocatechin gallate and
Poly E on the growth of various human colon cancer cell lines. The
x-axis of FIG. 2A represents the relative cell viability as a
percentage, while the y-axis represents the concentration of
(-)-epigallocatechin gallate in .mu.g/mL. The lines on the graph in
FIG. 2A from top to bottom, represent the following human colon
cancer cell lines: Caco2, HCT116, HT29, SW480, SW837 and FHC. The
x-axis of FIG. 2B represents the relative cell viability as a
percentage, while the y-axis represents the concentration of
(-)-epigallocatechin gallate in .mu.g/mL.
[0023] FIG. 3 shows a combination index isobologram which indicates
that (-)-epigallocatechin gallate and (-)-epicatechin exhibit a
synergistic effect on the growth of HT29 human colon cancer cells.
The y-axis represents the Combination Index, the rightmost x-axis
represents the concentration of (-)-epicatechin in .mu.g/mL, and
the leftmost x-axis represents the concentration of
(-)-epigallocatechin gallate in .mu.g/mL. A combination index of
1.1-1.3 is representative of moderate antagonism, a combination
index of 0.9-1.1 is representative of an additive effect, a
combination index of 0.8-0.9 is representative of slight synergism,
a combination index of 0.6-0.8 is representative of moderate
synergism, and a combination index of 0.4-0.6 is representative of
synergism.
[0024] FIG. 4 shows the sequence of oligonucleotide primers used
for PCR amplification for the IGFBP-3, IGF-1, MMP-7, MMP-9 and
TGF-.beta.2 genes.
[0025] FIG. 5 shows the expression levels of IGF-1.alpha.,
IGF-1.beta., p-IGF-1R, IGFBP-3, and IGF-1 proteins in the HCT116,
Caco2, HT29, SW837 and SW480 human colon cancer cell lines. Total
protein extracts were prepared from 70% confluent cultures of the
indicated cell lines and equivalent amounts of protein (60
.mu.g/lane) were examined by Western blot analysis using the
appropriate antibodies.
[0026] FIG. 6 shows the effects of EGCG on activation of the IGF-1R
and on levels of IGF-1 and IGFBP-3 proteins (FIGS. 6(a) and 6(b)),
and mRNAs (FIG. 6(c)) in SW837 human colon cancer cells. The cells
were treated with 20 mg/mL EGCG at 0, 3, 6, 12, 24 and 48 hours,
and cell extracts were examined by Western blot analysis using the
respective antibodies (FIG. 6(a)), or were examined by
semiquantitative RT-PCR analysis using IGFBP-3 or IGF-1 specific
primers (FIG. 6(c)). The results obtained from semiquantitative
RT-PCR analysis were quantitated by densitometry and are displayed
in the right panel of FIG. 6C. The effects of a low dose (10
.mu.g/mL) of EGCG on inhibition of the IGF-1R receptor activation
and on levels of IGF-1 and IGFBP-3 proteins are shown in FIG.
6(b).
[0027] FIG. 7 shows the effects of EGCG on MMPs-7 and 9 mRNAs (FIG.
7(a)) and on TGF-.beta.2 mRNA (FIG. 7(b)) in SW837 human colon
cancer cells. The cells were treated with 20 .mu.g/ML EGCG at 0, 3,
6, 12, 24 and 48 hours, and cell extracts were examined by
semiquantitative RT-PCR analysis using MMPs-7 and 9 (FIG. 7(a)),
and TGF-.beta.2 (FIG. 7(b)) specific primers. The results obtained
from semiquantitative RT-PCR analysis were quantitated by
densitometry and are displayed in the right panels of FIG. 7(a) and
FIG. 7(b). Amplified PCR products obtained with actin specific
primers served as internal controls.
[0028] FIG. 8 shows the expression levels of COX-2, HER3 and p-HER3
proteins in various colon cancer cell lines. Total protein extracts
were prepared from 70% confluent cultures of the indicated cell
lines and equivalent amounts of protein (60 .mu.g/lane) were
examined using western blot analysis for COX-2 (FIG. 8(a)), and
HER3 and p-HER3 (FIG. 8(b)), using the appropriate antibodies.
[0029] FIG. 9 shows the effect of EGCG on activation of the EGFR,
HER2, and HER3, and on related-downstream signaling pathways, and
on cellular levels of both COX-2 protein (FIG. 9(a)) and mRNA (FIG.
9(b)) in SW837 human colon cancer cells. The cells were treated
with 20 .mu.g/mL EGCG at 0, 3, 6, 12, 24 and 48 hours, and cell
extracts were examined by Western blot analysis using the
respective antibodies (FIG. 9(a)) or were examined by
semiquantitative RT-PCR analysis using COX-2 specific primers (FIG.
9(b)). An antibody to actin served as the loading control (FIG.
9(a)). Amplified PCR products obtained with actin specific primers
served as internal controls (FIG. 9(b)). The results of mRNA bands
were quantified by densitometry and these values were displayed as
fold expression (FIG. 9(b)). Similar results were obtained in a
repeat experiment. FIG. 9(c) shows the effects of EGCG on induction
of apoptosis in SW837 human colon cancer cells. The cells were
treated with 20 mg/mL EGCG or 0.1% DMSO for 48 hours, and cell
extracts were then examined for DNA fragmentation using the ELISA
DNA fragmentation system. The clear bars represent control cells
and the dark bars represent treated cells. An asterisk indicates a
significant difference (p<0.05) between the control
(DMSO-treated) cells and the EGCG-treated cells.
[0030] FIG. 10 shows the effects of EGCG on the transcriptional
activity of the following promoters in SW837 human colon cancer
cells: COX-2 (FIG. 10(a)), AP-1 (FIG. 10(b)), and NF-.kappa.B (FIG.
10(c)). Transient transfection reporter assays were performed with
the indicated luciferase reporter in the presence of the indicated
concentrations of EGCG. Relative luciferase activity was then
determined after 24 hours. An asterisk indicates a significant
difference (p<0.05) between the control (DMSO-treated) cells and
the EGCG-treated cells.
[0031] FIG. 11 shows the effects of EGCG on production of PGE2 by
SW837 human colon cancer cells. The cells were treated with the
indicated concentrations of EGCG for 18 hours in the presence of 20
mM arachadonic acid in serum-free medium. The cell-free medium was
then collected and assayed for released PGE2. An asterisk indicates
a significant difference (p<0.05) between the control
(DMSO-treated) cells and the EGCG-treated cells.
[0032] FIG. 12 shows the effects of a low dose of EGCG in SW837
human colon cancer cells. FIG. 12(a) shows the inhibition of growth
of SW837 human colon cancer cells. The cells were treated with EGCG
(1.0 .mu.g/mL or 20 .mu.g/mL) or 0.1% DMSO for 96 hours, and the
number of cells were then counted at the indicated times. FIG.
12(b) illustrates the induction of apoptosis in SW837 human colon
cancer cells. The cells were treated with 1.0 .mu.g/mL EGCG or 0.1%
DMSO for 96 hours, and cell extracts were examined for DNA
fragmentation using the ELISA system. An asterisk indicates a
significant difference (p<0.05) between the control
(DMSO-treated) cells and the EGCG-treated cells. FIG. 12(c) shows
the inhibition of the EGFR, HER2 and HER3 receptor activation in
SW837 human colon cancer cells and inhibition of cellular levels of
COX-3 and Bcl-x.sub.L proteins in SW837 human colon cancer cells.
The cells were treated with 1.0 .mu.g/mL EGCG or 0.1% DMSO for 72
hours or 96 hours, and cell extracts were examined using western
blot analysis using the respective antibodies. An antibody to actin
was used as the loading control. The clear bars represent control
cells and the dark bars represent treated cells. An asterisk
indicates a significant difference (p<0.05) between the control
(DMSO-treated) cells and the EGCG-treated cells.
[0033] FIG. 13 shows the effects of EGCG on levels of IGF-1, IGF-2
and IGFBP-3 proteins in HepG2 human liver cancer cells. The cells
were treated with 20 mg/mL EGCG at 0, 3, 6, 12, 24 and 48 hours,
and cell extracts were examined by Western blot analysis using the
respective antibodies, or were examined by semiquantitative RT-PCR
analysis using IGFBP-3 or IGF-1 specific primers. The results, set
forth in FIG. 13, show that EGCG decreased the production of IGF-1
and IGF-2 and increased the production of IGFBP-3.An asterisk
indicates a significant difference (p<0.05) between the control
(DMSO-treated) cells and the EGCG-treated cells.
5. DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention relates to Synergistic Polyphenol
Compounds, compositions thereof and methods for treating and
preventing cancer in a subject, the methods comprising
administering to a subject an effective amount of a Synergistic
Polyphenol Compound or composition thereof.
5.1 Definitions
[0035] The terms used herein having following meaning:
[0036] A "subject" is a mammal, e.g., a human, mouse, rat, guinea
pig, dog, cat, horse, cow, pig, or non-human primate, such as a
monkey, chimpanzee or baboon. In one embodiment, a monkey is a
rhesus. In another embodiment, a subject is a human.
[0037] The term "Synergistic Polyphenol Compound" refers to a
polyphenol compound or pharmaceutically acceptable salt thereof,
wherein the compound demonstrates a synergistic or additive effect
with: (a) one or more additional Synergistic Polyphenol Compounds
or (b) one or more other anticancer agents, for the treatment or
prevention of cancer in a subject. Illustrative Synergistic
Polyphenol Compounds include, but are not limited to, a catechin,
including but not limited to (-)-epicatechin, (-)-epicatechin
gallate, (-)-gallocatechin gallate, (-)-epigallocatechin and
(-)-epigallocatechin gallate; a phenolic acid, including but not
limited to gallic acid, caffeic acid and ellagic acid; a
bioflavanoid, including but not limited to an anthocyanin,
apigenin, and quercetin; and a complex polyphenol, including but
not limited to, a tannin and a lignan, and any combination thereof.
In one embodiment, a Synergistic Polyphenol Compound demonstrates a
synergistic effect with: (a) one or more additional Synergistic
Polyphenol Compounds and/or (b) one or more other anticancer
agents, for the treatment or prevention of cancer in a subject. In
one embodiment, a Synergistic Polyphenol Compound demonstrates an
additive effect with: (a) one or more additional Synergistic
Polyphenol Compounds and/or (b) one or more other anticancer
agents, for the treatment or prevention of cancer in a subject.
[0038] The term "Additive" when used in connection with the
Synergistic Polyphenol Compounds, means that the overall
therapeutic effect of a combination of: (a) two or more Synergistic
Polyphenol Compounds or (b) one or more Synergistic Polyphenol
Compounds and one or more other anticancer agents, when
administered as combination therapy for the treatment of cancer, is
equal to the sum of the therapeutic effects of these agents when
each is adminstered alone as monotherapy.
[0039] The term "Synergistic" when used in connection with the
Synergistic Polyphenol Compounds, means that the overall
therapeutic effect of a combination of: (a) two or more Synergistic
Polyphenol Compounds or (b) one or more Synergistic Polyphenol
Compounds and one or more other anticancer agents, when
administered as combination therapy for the treatment of cancer, is
greater than the sum of the therapeutic effects of these agents
when each is adminstered alone as monotherapy.
[0040] The term "Synergistic Polyphenol Composition" refers to a
composition comprising two or more Synergistic Polyphenol Compounds
or pharmaceutically acceptable salts thereof, and a physiologically
acceptable carrier or vehicle.
[0041] The term "in isolated form" as used herein means separated
from other components of a reaction mixture or natural source. In
certain embodiments, an isolate contains at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, or at least 98% of a
Synergistic Polyphenol Compound by weight of the isolate.
[0042] The phrase "consisting essentially of" as used herein in
connection with the identity of a Synergistic Polyphenol
Composition means that the Synergistic Polyphenol Composition
contains only those Synergistic Polyphenol Compounds that are set
forth as components of the Synergistic Polyphenol Composition. It
is to be understood that a Synergistic Polyphenol Composition that
consists essentially of specifically named Synergistic Polyphenol
Compounds may contain other materials in addition to the named
Synergistic Polyphenol Compounds, such that the other materials do
not materially affect the basic and novel characteristic(s) of the
Synergistic Polyphenol Composition.
[0043] The term "Poly E" refers to polyphenon E (Mitsui-Norin,
Ltd., Halifax, Canada), which is a standardized, commercially
available green tea extract and contains (-)-epigallocatechin
gallate, (-)-epigallocatechin, (-)-epicatechin gallate,
(-)-epicatechin and (-)-gallocatechin gallate.
[0044] The phrase "pharmaceutically acceptable salt," as used
herein, is a salt formed from an acid and a basic nitrogen group of
a Synergistic Polyphenol Compound. Illustrative salts include, but
are not limited, to sulfate, citrate, acetate, oxalate, chloride,
bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-OH-3-naphthoate)) salts. The term
"pharmaceutically acceptable salt" also refers to a salt prepared
from a Synergistic Polyphenol Compound having an acidic functional
group, such as a carboxylic acid functional group, and a
pharmaceutically acceptable inorganic or organic base. Suitable
bases include, but are not limited to, hydroxides of alkali metals
such as sodium, potassium, and lithium; hydroxides of alkaline
earth metal such as calcium and magnesium; hydroxides of other
metals, such as aluminum and zinc; ammonia, and organic amines,
such as unsubstituted or hydroxy-substituted mono-, di-, or
tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine;
N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-,
or tris-(2-hydroxy substituted lower alkylamines), such as mono-;
bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or
tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy
lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine
or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids
such as arginine, lysine, and the like. The term "pharmaceutically
acceptable salt" also includes a hydrate of a Synergistic
Polyphenol Compound.
[0045] The following abbreviations are used herein and have the
following meanings: COX-2 is cyclooxygenase 2; DMSO is
N,N-dimethylsulfoxide; EGCG is epigallocatechin gallate; EGFR is
epidermal growth factor receptor; ERK is extracellular related
kinase; HER2 is herceptin 2; HER3 is herceptin 3; IGF-1 is
insulin-like growth factor 1; IGF-1 R is insulin-like growth factor
1 receptor; IGFBP-3 is insulin-like growth factor binding protein
3; PGE.sub.2 is prostaglandin E.sub.2; PBS is phosphate-buffered
saline; RTK is receptor tyrosine kinase; and RT-PCR is reverse
transcriptase polymerase chain reaction.
5.2 The Synergistic Polyphenol Compounds
[0046] As stated above, the present invention encompasses methods
for treating or preventing cancer in a subject, the methods
comprising administering to the subject: (i) an effective amount of
a Synergistic Polyphenol Composition or (ii) two or more
Synergistic Polyphenol Compounds wherein the amounts of the two or
more Synergistic Polyphenol Compounds are together synergistically
effective to treat or prevent cancer.
[0047] Illustrative Synergistic Polyphenol Compounds useful in the
Synergistic Polyphenol Compositions and present methods for
treating or preventing cancer, include, but are not limited to the
following compounds and pharmaceutically acceptable salts thereof:
a catechin, including but not limited to (-)-epicatechin,
(-)-epicatechin gallate, (-)-gallocatechin gallate,
(-)-epigallocatechin and (-)-epigallocatechin gallate; a phenolic
acid, including but not limited to gallic acid, caffeic acid and
ellagic acid; a bioflavanoid, including but not limited to an
anthocyanin, apigenin, and quercetin; and a complex polyphenol,
including but not limited to, a tannin and a lignan, and any
combination thereof.
[0048] In one embodiment, the Synergistic Polyphenol Compound is
(-)-epigallocatechin gallate.
[0049] In another embodiment, the Synergistic Polyphenol Compound
is (+)-polyphenol.
[0050] In another embodiment, the Synergistic Polyphenol Compound
is (-)-epicatechin.
[0051] In still another embodiment, the Synergistic Polyphenol
Compound is (-)-epicatechin gallate.
[0052] In yet another embodiment, the Synergistic Polyphenol
Compound is (-)-gallocatechin gallate.
[0053] In a further embodiment, the Synergistic Polyphenol Compound
is (-)-epigallocatechin.
[0054] In one embodiment, the Synergistic Polyphenol Compound is a
bioflavanoid.
[0055] In another embodiment, the Synergistic Polyphenol Compound
is a phenolic acid.
[0056] In another embodiment, the Synergistic Polyphenol Compound
is a tannin.
[0057] In still another embodiment, the Synergistic Polyphenol
Compound is a lignan.
[0058] The Synergistic Polyphenol Compounds may be purchased from
commercial sources (e.g., Sigma Chemical, St. Louis, Mo.), prepared
synthetically using methods well-known to one skilled in the art of
synthetic organic chemistry, or extracted from natural sources
using methods well-known to one skilled in the arts of chemistry
and/or biology and/or related arts.
[0059] It is possible for some of the Synergistic Polyphenol
Compounds to have one or more chiral centers and as such these
Synergistic Polyphenol Compounds can exist in various
stereoisomeric forms. Accordingly, the present invention is
understood to encompass all possible stereoisomers.
[0060] In one embodiment, a Synergistic Polyphenol Compound is
obtained from a natural product extract. In a specific embodiment,
a Synergistic Polyphenol Compound is obtained from a green tea
extract.
[0061] In another embodiment, a Synergistic Polyphenol Compound is
in isolated form.
5.3 The Synergistic Polyphenol Compositions
[0062] In one embodiment, the Synergistic Polyphenol Compositions
comprise two or more Synergistic Polyphenol Compounds and a
physiologically acceptable carrier or vehicle, and are useful for
treating or preventing cancer in a subject.
[0063] In one embodiment, a Synergistic Polyphenol Composition
comprises (-)-epigallocatechin gallate.
[0064] In another embodiment, a Synergistic Polyphenol Composition
comprises (+)-polyphenol.
[0065] In another embodiment, a Synergistic Polyphenol Composition
comprises (-)-epicatechin.
[0066] In still another embodiment, a Synergistic Polyphenol
Composition comprises (-)-epicatechin gallate.
[0067] In yet another embodiment, a Synergistic Polyphenol
Composition comprises (-)-gallocatechin gallate.
[0068] In a further embodiment, a Synergistic Polyphenol
Composition comprises (-)-epigallocatechin.
[0069] In one embodiment, a Synergistic Polyphenol Composition
comprises a bioflavanoid.
[0070] In another embodiment, a Synergistic Polyphenol Composition
comprises a phenolic acid.
[0071] In another embodiment, a Synergistic Polyphenol Composition
comprises a tannin.
[0072] In still another embodiment, a Synergistic Polyphenol
Composition comprises a lignan.
[0073] In one embodiment, a Synergistic Polyphenol Composition
comprises (-)-epigallocatechin gallate and (-)-epicatechin.
[0074] In another embodiment, a Synergistic Polyphenol Composition
comprises (-)-epigallocatechin gallate and (+)-polyphenol.
[0075] In another embodiment, a Synergistic Polyphenol Composition
comprises (-)-epigallocatechin gallate and (-)-epicatechin
gallate.
[0076] In still another embodiment, a Synergistic Polyphenol
Composition comprises (-)-epigallocatechin gallate and
(-)-gallocatechin gallate.
[0077] In a further embodiment, a Synergistic Polyphenol
Composition comprises (-)-epigallocatechin gallate and
(-)-epigallocatechin.
[0078] In one embodiment, a Synergistic Polyphenol Composition
comprises (-)-epigallocatechin gallate, (-)-epicatechin,
(-)-epicatechin gallate, (-)-gallocatechin gallate and
(-)-epigallocatechin.
[0079] In one embodiment, a Synergistic Polyphenol Composition
comprises (-)-epigallocatechin gallate and epicatechin such that
the ratio of (-)-epigallocatechin gallate to (-)-epicatechin by
weight is from (a) about 20 to about 1 to 10 to (b) about 1 to
about 10.
[0080] In various embodiments, a Synergistic Polyphenol Composition
comprises (-)-epigallocatechin gallate and epicatechin such that
the ratio of (-)-epigallocatechin gallate to (-)-epicatechin by
weight is about 20 to about 1; about 10 to about 1; about 2 to
about 1; about 1 to about 1; about 2 to about 5, about 1 to about
5, or about 1 to about 10.
[0081] In one embodiment, the Synergistic Polyphenol Compositions
consist essentially of two or more Synergistic Polyphenol Compounds
and a physiologically acceptable carrier or vehicle, and are useful
for treating or preventing cancer in a subject.
[0082] In one embodiment, a Synergistic Polyphenol Composition
consists essentially of (-)-epigallocatechin gallate and
(-)-epicatechin.
[0083] In another embodiment, a Synergistic Polyphenol Composition
consists essentially of (-)-epigallocatechin gallate and
(+)-polyphenol.
[0084] In another embodiment, a Synergistic Polyphenol Composition
consists essentially of (-)-epigallocatechin gallate and
(-)-epicatechin gallate.
[0085] In still another embodiment, a Synergistic Polyphenol
Composition consists essentially of (-)-epigallocatechin gallate
and (-)-gallocatechin gallate.
[0086] In a further embodiment, a Synergistic Polyphenol
Composition consists essentially of (-)-epigallocatechin gallate
and (-)-epigallocatechin.
[0087] In one embodiment, a Synergistic Polyphenol Composition
consists essentially of (-)-epigallocatechin gallate,
(-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin gallate
and (-)-epigallocatechin.
[0088] In one embodiment, a Synergistic Polyphenol Composition
consists essentially of (-)-epigallocatechin gallate and
epicatechin such that the ratio of (-)-epigallocatechin gallate to
(-)-epicatechin by weight is from (a) about 20 to about 1 to 10 to
(b) about 1 to about 10.
[0089] In various embodiments, a Synergistic Polyphenol Composition
consists essentially of (-)-epigallocatechin gallate and
(-)-epicatechin such that the ratio of (-)-epigallocatechin gallate
to (-)-epicatechin by weight is about 20 to about 1; about 10 to
about 1; about 2 to about 1; about 1 to about 1; about 2 to about
5, about 1 to about 5, or about 1 to about 10.
[0090] In one embodiment, one or more of the Synergistic Polyphenol
Compounds of a Synergistic Polyphenol Composition is in isolated
form.
5.3.1 Obtaining the Synergistic Polyphenol Compositions
[0091] The Synergistic Polyphenol Compositions can be prepared
using combinations of various isolated or crude Synergistic
Polyphenol Compounds or may be obtained as extracts from natural
products, such as green tea.
5.3.1.1 Green Tea Extracts
[0092] Green tea leaves have been found to contain numerous
biologically active compounds that are potentially useful in
medical and veterinary applications, including various members of
the polyphenol family of natural products. As such, in one
embodiment, a Synergistic Polyphenol Composition can be obtained
from green tea leaves using extraction procedures well known to one
skilled in the relevant art. The extraction procedures may be
carried out using water, polar organic solvents, non-polar organic
solvents, supercritical fluids, or mixtures thereof. Organic
solvents useful for extracting a Synergistic Polyphenol Composition
from green tea leaves include, but are not limited to alcohols,
such as methanol, ethanol, n-propanol, isopropanol, n-butanol,
isobutanol, sec-butanol and tert-butanol; ketones, such as acetone,
methyl ethyl ketone, and ethyl acetate; ethers, such as diethyl
ether, diphenyl ether, tetrahydrofuran, and dioxane; aliphatic
hydrocarbons, such as pentanes, hexanes, and heptanes; aromatic
hydrocarbons such as benzene, toluene, naphthalene, and xylenes;
alkyl halides, such as carbon tetrachloride, choroform and
methylene chloride; amides, such as dimethylformamide and
hexamethylphosporamide; carboxylic acids, such as formic acid and
acetic acid; and sulfoxides, such as dimethylsulfoxide.
[0093] As stated above, the present invention encompasses methods
for treating or preventing cancer in a subject, the methods
comprising administering to the subject an effective amount of a
Synergistic Polyphenol Composition extracted from green tea leaves.
Green tea extracts containing a Synergistic Polyphenol Composition
include both oil and water soluble extracts and can be obtained
from commercial sources (e.g., Nature's Resource, Mission Hills,
Calif., or Herbasin, Beijing, China) or can be obtained directly
from green tea leaves using known extraction methods, such as those
disclosed in European Patent No. EP 1402869 to Schneider, which is
hereby incorporated by reference in its entirety.
[0094] In one embodiment, the Synergistic Polyphenol Composition is
Poly E.
[0095] In another embodiment, the Synergistic Polyphenol
Composition is extracted from a natural source.
5.4 Uses of the Compounds and Compositions of the Invention
5.4.1 Treatment or Prevention of Cancer
[0096] The Synergistic Polyphenol Compounds or Synergistic
Polyphenol Compositions are useful for treating or preventing
cancer.
[0097] In one embodiment, the invention provides a method for
treating cancer in a subject, the method comprising:
[0098] (a) administering to the subject a first Synergistic
Polyphenol Compound; and
[0099] (b) administering to the subject a second Synergistic
Polyphenol Compound, wherein the first and second compounds are
different and wherein the amounts of the first and second compounds
administered are together synergistically effective to treat or
prevent cancer.
[0100] In another embodiment, the invention provides a method for
treating colorectal cancer or liver cancer in a subject, the method
comprising:
[0101] (a) administering to the subject a first Synergistic
Polyphenol Compound; and
[0102] (b) administering to the subject a second Synergistic
Polyphenol Compound, wherein the first and second compounds are
different and wherein the amounts of the first and second compounds
administered are together synergistically effective to treat or
prevent colorectal cancer or liver cancer.
[0103] In one embodiment, the cancer being treated or prevented is
colorectal cancer.
[0104] In another embodiment, the cancer being treated or prevented
is liver cancer.
[0105] In one embodiment, the first compound is
(-)-epigallocatechin gallate.
[0106] In another embodiment the first compound is
(-)-epicatechin.
[0107] In another embodiment, the first compound is
(-)-epigallocatechin gallate and the second compound is
(-)-epicatechin.
[0108] In still another embodiment, the first compound is
(-)-epigallocatechin gallate and the second compound is
(-)-epicatechin, wherein the ratio of the amount of (-)-epicatechin
administered to the amount of (-)-epigallocatechin gallate
administered to is about 5 to about 2.
[0109] In a further embodiment, the first compound is
(-)-epigallocatechin gallate and the second compound is
(-)-epicatechin, wherein the ratio of the amount of (-)-epicatechin
administered to the amount of (-)-epigallocatechin gallate
administered to is about 5 to about 1.
[0110] In various embodiments, the first compound is
(-)-epigallocatechin gallate and the second compound is
(-)-epicatechin such that the ratio of (-)-epigallocatechin gallate
administered to the amount of (-)-epicatechin administered is about
20 to about 1; about 10 to about 1; about 2 to about 1; about 1 to
about 1; about 2 to about 5, about 1 to about 5, or about 1 to
about 10.
[0111] In another embodiment, the invention provides a method for
treating cancer in a subject, the method comprising administering
to the subject an effective amount of a Synergistic Polyphenol
Composition.
[0112] Examples of cancers treatable or preventable using the
Synergistic Polyphenol Compounds or Synergistic Polyphenol
Compositions include, but are not limited to, the cancers disclosed
below in Table 1 and metastases thereof. TABLE-US-00001 TABLE 1
Solid tumors, including but not limited to: fibrosarcoma
myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma
angiosarcoma endotheliosarcoma lymphangiosarcoma
lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor
leiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer
kidney cancer pancreatic cancer bone cancer breast cancer ovarian
cancer prostate cancer esophageal cancer stomach cancer oral cancer
nasal cancer throat cancer squamous cell carcinoma basal cell
carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland
carcinoma papillary carcinoma papillary adenocarcinomas
cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal
cell carcinoma hepatoma liver cancer bile duct carcinoma
choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical
cancer uterine cancer testicular cancer small cell lung carcinoma
bladder carcinoma lung cancer epithelial carcinoma skin cancer
melanoma neuroblastoma retinoblastoma blood-borne cancers,
including but not limited to: acute lymphoblastic leukemia ("ALL")
acute lymphoblastic B-cell leukemia acute lymphoblastic T-cell
leukemia acute myeloblastic leukemia ("AML") acute promyelocytic
leukemia ("APL") acute monoblastic leukemia acute erythroleukemic
leukemia acute megakaryoblastic leukemia acute myelomonocytic
leukemia acute nonlymphocyctic leukemia acute undifferentiated
leukemia chronic myelocytic leukemia ("CML") chronic lymphocytic
leukemia ("CLL") hairy cell leukemia multiple myeloma acute and
chronic leukemias: lymphoblastic myelogenous lymphocytic myelocytic
leukemias Lymphomas: Hodgkin's disease non-Hodgkin's Lymphoma
Multiple myeloma Waldenstrom's macroglobulinemia Heavy chain
disease Polycythemia vera CNS and brain cancers: glioma pilocytic
astrocytoma astrocytoma anaplastic astrocytoma glioblastoma
multiforme medulloblastoma craniopharyngioma ependymoma pinealoma
hemangioblastoma acoustic neuroma oligodendroglioma meningioma
vestibular schwannoma adenoma metastatic brain tumor meningioma
spinal tumor medulloblastoma
[0113] In one embodiment the cancer is lung cancer, breast cancer,
colorectal cancer, prostate cancer, a leukemia, a lymphoma, a
non-Hodgkin's lymphoma, a skin cancer, a brain cancer, a cancer of
the central nervous system, ovarian cancer, uterine cancer, stomach
cancer, pancreatic cancer, esophageal cancer, kidney cancer, liver
cancer, or a head and neck cancer.
[0114] In one embodiment, the cancer is a solid tumor.
[0115] In another embodiment, the cancer is a systemic cancer, such
as a leukemia or a lymphoma.
[0116] In a specific embodiment, the cancer is colorectal
cancer.
[0117] In another specific embodiment the cancer is breast
cancer.
[0118] In another specific embodiment the cancer is liver
cancer.
[0119] In one embodiment, the subject has previously undergone or
is presently undergoing treatment for cancer. Such previous
treatments include, but are not limited to, prior chemotherapy,
radiation therapy, surgery, or immunotherapy, such as a cancer
vaccine.
[0120] The Synergistic Polyphenol Compounds or Synergistic
Polyphenol Compositions are also useful for the treatment or
prevention of a cancer caused by a virus. Such viruses include
human papilloma virus, which can lead to cervical cancer (see,
e.g., Hernandez-Avila et al., Archives of Medical Research (1997)
28:265-271); Epstein-Barr virus (EBV), which can lead to lymphoma
(see, e.g., Herrmann et al., J Pathol (2003) 199(2):140-5);
hepatitis B or C virus, which can lead to liver carcinoma (see,
e.g., E1-Serag, J Clin Gastroenterol (2002) 35(5 Suppl 2):S72-8);
human T cell leukemia virus (HTLV)-I, which can lead to T-cell
leukemia (see e.g., Mortreux et al., Leukemia (2003) 17(1):26-38);
human herpesvirus-8 infection, which can lead to Kaposi's sarcoma
(see, e.g., Kadow et al., Curr Opin Investig Drugs (2002)
3(11):1574-9); and Human Immune deficiency Virus (HIV) infection,
which can lead to cancer as a consequence of immunodeficiency (see,
e.g., Dal Maso et al., Lancet Oncol (2003) 4(2):110-9).
[0121] The Synergistic Polyphenol Compounds or Synergistic
Polyphenol Compositions can also be administered to prevent the
progression of a cancer, including but not limited to the cancers
listed in Table 1. Such prophylactic use includes that in which
non-neoplastic cell growth consisting of hyperplasia, metaplasia,
or most particularly, dysplasia has occurred.
[0122] Alternatively or in addition to the presence of abnormal
cell growth characterized as hyperplasia, metaplasia, or dysplasia,
the presence of one or more characteristics of a transformed
phenotype, or of a malignant phenotype, displayed in vivo or
displayed in vitro by a cell sample from an animal, can indicate
the desirability of prophylactic/therapeutic administration of the
Synergistic Polyphenol Compounds or Synergistic Polyphenol
Compositions. Such characteristics of a transformed phenotype
include morphology changes, looser substratum attachment, loss of
contact inhibition, loss of anchorage dependence, protease release,
increased sugar transport, decreased serum requirement, expression
of fetal antigens, disappearance of the 250,000 dalton cell surface
protein, etc. (see also id., at pp. 84-90 for characteristics
associated with a transformed or malignant phenotype).
[0123] In a specific embodiment, leukoplakia, a benign-appearing
hyperplastic or dysplastic lesion of the epithelium, or Bowen's
disease, a carcinoma in situ, are treatable or preventable
according to the present methods.
[0124] In another embodiment, fibrocystic disease (cystic
hyperplasia, mammary dysplasia, particularly adenosis (benign
epithelial hyperplasia)) are treatable or preventable according to
the present methods.
[0125] In other embodiments, a subject that exhibits one or more of
the following predisposing factors for malignancy can be
administered an amount of two or more Synergistic Polyphenol
Compounds or an amount of a Synergistic Polyphenol Composition
which is effective to treat or prevent cancer: a chromosomal
translocation associated with a malignancy (e.g., the Philadelphia
chromosome for chronic myelogenous leukemia, t(14;18) for
follicular lymphoma); familial polyposis or Gardner's syndrome;
benign monoclonal gammopathy; a first degree kinship with persons
having a cancer or precancerous disease showing a Mendelian
(genetic) inheritance pattern (e.g., familial polyposis of the
colorectal, Gardner's syndrome, hereditary exostosis, polyendocrine
adenomatosis, medullary thyroid carcinoma with amyloid production
and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of
Von Recklinghausen, retinoblastoma, carotid body tumor, cutaneous
melanocarcinoma, intraocular melanocarcinoma, xeroderma
pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome,
albinism, Fanconi's aplastic anemia, and Bloom's syndrome; and
exposure to carcinogens (e.g., smoking, second-hand smoke exposure,
and inhalation of or contacting with certain chemicals).
5.4.2 Combination Chemotherapy for the Treatment of Cancer
[0126] In one embodiment, the present methods for treating cancer
or preventing cancer further comprise administering another
anticancer agent that is not a Synergistic Polyphenol Compound.
[0127] In one embodiment, the present invention provides methods
for treating or preventing cancer in a subject, the method
comprising the administration of an effective amount of: (i) a
Synergistic Polyphenol Composition or two or more Synergistic
Polyphenol Compounds, and (ii) another anticancer agent.
[0128] In one embodiment, (i) a Synergistic Polyphenol Composition
or two or more Synergistic Polyphenol Compounds, and (ii) another
anticancer agent are administered in doses commonly employed when
such agents are used alone for the treatment of cancer.
[0129] In another embodiment, (i) a Synergistic Polyphenol
Composition or two or more Synergistic Polyphenol Compounds, and
(ii) another anticancer agent act synergistically and are
administered in doses that are less than the doses commonly
employed when such agents are used alone for the treatment of
cancer.
[0130] The dosage of the (i) a Synergistic Polyphenol Composition
or two or more Synergistic Polyphenol Compounds, and (ii) another
anticancer agent administered as well as the dosing schedule can
depend on various parameters, including, but not limited to, the
cancer being treated, the subject's general health, and the
administering physician's discretion.
[0131] A Synergistic Polyphenol Composition or Synergistic
Polyphenol Compound can be administered prior to (e.g., 5 minutes,
15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6
hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2
weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks
before), concurrently with, or subsequent to (e.g., 5 minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,
3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of the other anticancer agent to a subject in need
thereof. In various embodiments, i) a Synergistic Polyphenol
Composition or two or more Synergistic Polyphenol Compounds, and
(ii) another anticancer agent are administered 1 minuteute apart,
10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour
to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours
apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours
to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours
apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11
hours to 12 hours apart, no more than 24 hours apart, or no more
than 48 hours apart. In one embodiment, i) a Synergistic Polyphenol
Composition and (ii) another anticancer agent are administered with
3 hours. In another embodiment, i) a Synergistic Polyphenol
Composition and (ii) another anticancer agent are administered 1
minuteute to 24 hours apart.
[0132] In one embodiment, a Synergistic Polyphenol Composition
further comprises an effective amount of another anticancer agent,
such that the Synergistic Polyphenol Compounds of the Synergistic
Polyphenol Composition and the other anticancer agent are present
in the same composition. In one embodiment, this composition is
useful for oral administration. In another embodiment, this
composition is useful for intravenous administration.
[0133] Cancers that can be treated or prevented by administering
and effective amount of (i) a Synergistic Polyphenol Composition or
two or more Synergistic Polyphenol Compounds, and (ii) another
anticancer agent include, but are not limited to, the list of
cancers set forth in Table 1.
[0134] In one embodiment the cancer is lung cancer, breast cancer,
colorectal cancer, prostate cancer, a leukemia, a lymphoma, a
non-Hodgkin's lymphoma, a skin cancer, a brain cancer, a cancer of
the central nervous system, ovarian cancer, uterine cancer, stomach
cancer, pancreatic cancer, esophageal cancer, kidney cancer, liver
cancer, or a head and neck cancer.
[0135] In another embodiment, the cancer is colorectal cancer.
[0136] In still another embodiment the cancer is breast cancer.
[0137] In another embodiment the cancer is liver cancer.
[0138] The (i) Synergistic Polyphenol Composition or two or more
Synergistic Polyphenol Compounds, and (ii) other anticancer agent,
can act additively or synergistically. A synergistic combination of
the (i) Synergistic Polyphenol Composition or two or more
Synergistic Polyphenol Compounds, and (ii) other anticancer agent
might allow the use of lower dosages of the (i) Synergistic
Polyphenol Composition or two or more Synergistic Polyphenol
Compounds, and/or (ii) other anticancer agent and/or less frequent
dosages of the (i) Synergistic Polyphenol Composition or two or
more Synergistic Polyphenol Compounds, and (ii) other anticancer
agent, and/or to administer the (i) Synergistic Polyphenol
Composition or two or more Synergistic Polyphenol Compounds, and/or
(ii) other anticancer agent less frequently can reduce any toxicity
associated with the administration of (i) and/or (ii) to a subject
without reducing the efficacy of (i) and/or (ii) in the treatment
of cancer. In addition, a synergistic effect might result in the
improved efficacy of these agents in the treatment of cancer and/or
the reduction of any adverse or unwanted side effects associated
with the use of either agent alone.
[0139] In one embodiment, a (i) Synergistic Polyphenol Composition
or a Synergistic Polyphenol Compound, and (ii) another anticancer
agent act synergistically when administered in doses typically
employed when such agents are used alone for the treatment of
cancer. In another embodiment, a (i) Synergistic Polyphenol
Composition or a Synergistic Polyphenol Compound, and (ii) another
anticancer agent, act synergistically when administered in doses
that are less than doses typically employed when such agents are
used alone for the treatment of cancer.
[0140] In one embodiment, the administration of an effective amount
of a (i) Synergistic Polyphenol Composition or a Synergistic
Polyphenol Compound, and (ii) another anticancer agent inhibits the
resistance of a cancer to the other anticancer agent.
[0141] Suitable other anticancer agents useful in the methods and
compositions of the present invention include, but are not limited
to temozolomide, a topoisomerase I inhibitor, procarbazine,
dacarbazine, gemcitabine, capecitabine, methotrexate, taxol,
taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, etoposide, teniposide,
campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin,
dactinomycin, plicamycin, mitoxantrone, L-asparaginase,
doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel
and paclitaxel, leucovorin, levamisole, irinotecan, estramustine,
etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustine
and lomustine, vinca alkaloids such as vinblastine, vincristine and
vinorelbine, platinum complexes such as cisplatin, carboplatin and
oxaliplatin, imatinib mesylate, hexamethylmelamine, topotecan,
tyrosine kinase inhibitors, tyrphostins herbimycin A, genistein,
erbstatin, and lavendustin A.
[0142] In one embodiment, the other anticancer agents useful in the
methods and compositions of the present invention include, but are
not limited to, a drug listed in Table 2 or a pharmaceutically
acceptable salt thereof. TABLE-US-00002 TABLE 2 Alkylating agents
Nitrogen mustards: Cyclophosphamide Ifosfamide Trofosfamide
Chlorambucil Nitrosoureas: Carmustine (BCNU) Lomustine (CCNU)
Alkylsulphonates: Busulfan Treosulfan Triazenes: Dacarbazine
Procarbazine Temozolomide Platinum containing complexes: Cisplatin
Carboplatin Aroplatin Oxaliplatin Plant Alkaloids Vinca alkaloids:
Vincristine Vinblastine Vindesine Vinorelbine Taxoids: Paclitaxel
Docetaxel DNA Topoisomerase Inhibitors Epipodophyllins: Etoposide
Teniposide Topotecan Irinotecan 9-aminocamptothecin Camptothecin
Crisnatol Mitomycins: Mitomycin C Anti-metabolites Anti-folates:
DHFR inhibitors: Methotrexate Trimetrexate IMP dehydrogenase
Inhibitors: Mycophenolic acid Tiazofurin Ribavirin EICAR
Ribonuclotide reductase Hydroxyurea Inhibitors: Deferoxamine
Pyrimidine analogs: Uracil analogs: 5-Fluorouracil Fluoxuridine
Doxifluridine Ralitrexed Cytosine analogs: Cytarabine (ara C)
Cytosine arabinoside Fludarabine Gemcitabine Capecitabine Purine
analogs: Mercaptopurine Thioguanine O-6-benzylguanine DNA
Antimetabolites: 3-HP 2'-deoxy-5-fluorouridine 5-HP alpha-TGDR
aphidicolin glycinate ara-C 5-aza-2'-deoxycytidine beta-TGDR
cyclocytidine guanazole inosine glycodialdehyde macebecin II
Pyrazoloimidazole Hormonal therapies: Receptor antagonists:
Anti-estrogen: Tamoxifen Raloxifene Megestrol LHRH agonists:
Goscrclin Leuprolide acetate Anti-androgens: Flutamide Bicalutamide
Retinoids/Deltoids Cis-retinoic acid Vitamin A derivative:
All-trans retinoic acid (ATRA-IV) Vitamin D3 analogs: EB 1089 CB
1093 KH 1060 Photodynamic therapies: Vertoporfin (BPD-MA)
Phthalocyanine Photosensitizer Pc4 Demethoxy-hypocrellin A
(2BA-2-DMHA) Cytokines: Interferon-.alpha. Interferon-.beta.
Interferon-.gamma. Tumor necrosis factor Interleukin-2 Angiogenesis
Inhibitors: Angiostatin (plasminogen fragment) antiangiogenic
antithrombin III Angiozyme ABT-627 Bay 12-9566 Benefin Bevacizumab
BMS-275291 cartilage-derived inhibitor (CDI) CAI CD59 complement
fragment CEP-7055 Col 3 Combretastatin A-4 Endostatin (collagen
XVIII fragment) Fibronectin fragment Gro-beta Halofuginone
Heparinases Heparin hexasaccharide fragment HMV833 Human chorionic
gonadotropin (hCG) IM-862 Interferon alpha/beta/gamma Interferon
inducible protein (IP- 10) Interleukin-12 Kringle 5 (plasminogen
fragment) Marimastat Metalloproteinase inhibitors (TIMPs)
2-Methoxyestradiol MMI 270 (CGS 27023A) MoAb IMC-1C11 Neovastat
NM-3 Panzem PI-88 Placental ribonuclease inhibitor Plasminogen
activator inhibitor Platelet factor-4 (PF4) Prinomastat Prolactin
16 kD fragment Proliferin-related protein (PRP) PTK 787/ZK 222594
Retinoids Solimastat Squalamine SS 3304 SU 5416 SU6668 SU11248
Tetrahydrocortisol-S Tetrathiomolybdate Thalidomide
Thrombospondin-1 (TSP-1) TNP-470 Transforming growth factor-beta
(TGF-b) Vasculostatin Vasostatin (calreticulin fragment) ZD6126 ZD
6474 farnesyl transferase inhibitors (FTI) Bisphosphonates
Antimitotic agents: Allocolchicine Halichondrin B Colchicine
colchicine derivative dolstatin 10 Maytansine Rhizoxin
Thiocolchicine trityl cysteine Others: Protein Kinase G inhibitors:
OSI 461 Exisulind Tyrosine Kinase inhibitors: Iressa Tarceva
Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell
cycle inhibitors: Staurosporine Actinomycins: Actinomycin D
Dactinomycin Bleomycins: Bleomycin A2 Bleomycin B2 Peplomycin
Anthracyclines: Daunorubicin Doxorubicin Idarubicin Epirubicin
Pirarubicin Zorubicin Mitoxantrone MDR inhibitors: Verapamil
Ca.sup.2+ ATPase inhibitors: Thapsigargin
[0143] In one embodiment, the other anticancer agent is OSI
461.
[0144] In another embodiment, the other anticancer agent is
Iressa.
[0145] In still another embodiment, the other anticancer agent is
taxol.
[0146] In a further embodiment, the other anticancer agent is
5-fluorouracil.
[0147] In yet another embodiment, the other anticancer agent is a
platinum-based anticancer agent.
[0148] In one embodiment, the platinum-based anticancer agent is
cisplatin, carboplatin or oxaliplatin.
5.4.3 Multi-Therapy for Cancer
[0149] A Synergistic Polyphenol Compound or Synergistic Polyphenol
Composition can be administered to a subject that has undergone or
is currently undergoing one or more additional anticancer therapies
including, but not limited to, surgery, radiation therapy, or
immunotherapy, such as cancer vaccines.
[0150] In one embodiment, the invention provides methods for
treating or preventing cancer comprising administering to a subject
in need thereof (a) an effective amount of a Synergistic Polyphenol
Composition or two or more Synergistic Polyphenol Compounds, and
(b) another anticancer therapy including, but not limited to,
surgery, radiation therapy, or immunotherapy, such as a cancer
vaccine.
[0151] In one embodiment, the other anticancer therapy is radiation
therapy.
[0152] In another embodiment, the other anticancer therapy is
surgery.
[0153] In still another embodiment, the other anticancer therapy is
immunotherapy.
[0154] In a specific embodiment, the present methods for treating
or preventing cancer comprise administering (i) a Synergistic
Polyphenol Composition or two or more Synergistic Polyphenol
Compounds, and (ii) radiation therapy. The radiation therapy can be
administered concurrently with, prior to, or subsequent to the
Synergistic Polyphenol Composition or two or more Synergistic
Polyphenol Compounds. In various embodiments, radiation therapy is
administered at least 30 minutes, one hour, five hours, 12 hours,
one day, one week, one month, or several months (e.g., up to three
months), prior or subsequent to administration of a Synergistic
Polyphenol Composition or Synergistic Polyphenol Compound.
[0155] Where the other anticancer therapy is radiation therapy, any
radiation therapy protocol can be used depending upon the type of
cancer to be treated. For example, but not by way of limitation,
X-ray radiation can be administered; in particular, high-energy
megavoltage (radiation of greater that 1 MeV energy) can be used
for deep tumors, and electron beam and orthovoltage X-ray radiation
can be used for skin cancers. Gamma-ray emitting radioisotopes,
such as radioactive isotopes of radium, cobalt and other elements,
can also be administered.
[0156] Additionally, in one embodiment the invention provides
methods of treatment of cancer using a Synergistic Polyphenol
Composition or two or more Synergistic Polyphenol Compounds as an
alternative to chemotherapy or radiation therapy where the
chemotherapy or the radiation therapy results in negative side
effects, in the subject being treated. The subject being treated
can, optionally, be treated with another anticancer therapy such as
surgery, radiation therapy, or immunotherapy.
[0157] The Synergistic Polyphenol Compositions and Synergistic
Polyphenol Compounds can also be used in vitro or ex vivo, such as
for the treatment of certain cancers, including, but not limited to
leukemias and lymphomas, such treatment involving autologous stem
cell transplants. This can involve a process in which the subject's
autologous hematopoietic stem cells are harvested and purged of all
cancer cells, the subject's remaining bone-marrow cell population
is then eradicated via the administration of (i) a Synergistic
Polyphenol Composition or two or more Synergistic Polyphenol
Compounds, and/or (ii) radiation, and the resultant stem cells are
then infused back into the subject. Supportive care can be
subsequently provided while bone marrow function is restored and
the subject recovers.
5.5 Therapeutic/Prophylactic Administration and Compositions of the
Invention
[0158] Due to their activity, the Synergistic Polyphenol Compounds
or Synergistic Polyphenol Compositions are advantageously useful in
veterinary and human medicine. As described above, the Synergistic
Polyphenol Compounds or Synergistic Polyphenol Compositions are
useful for treating or preventing cancer in a subject in need
thereof.
[0159] A Synergistic Polyphenol Composition comprises synergistic
amounts of two or more Synergistic Polyphenol Compounds. In one
embodiment, a Synergistic Polyphenol Composition may contain: (a)
an amount of a first Synergistic Polyphenol Compound which is less
than the amount of said Compound when said Compound is administered
as a single-agent, (b) an amount of a second Synergistic Polyphenol
Compound which is less than the amount of said Compound when said
Compound is administered as a single-agent, and (c) an amount of
one or more additional anticancer agents or pharmaceutically
acceptable salts thereof, which is less than the amount of said
additional anticancer agents when said additional anticancer agent
is administered as a single-agent. In another embodiment, a
synergistic combination may contain an amount of a first
Synergistic Polyphenol Compound, an amount of a second Synergistic
Polyphenol Compound, and an amount of one or more additional
anticancer agents or pharmaceutically acceptable salts thereof,
which is similar to the amounts used when each of these agents are
administered alone for the treatment of cancer.
[0160] The Synergistic Polyphenol Compositions of the present
invention can be in any form that allows for the composition to be
administered to a subject.
[0161] When administered to a subject, a Synergistic Polyphenol
Compound can be administered as a component of a composition that
comprises a physiologically acceptable carrier or vehicle. In one
embodiment, the composition further comprises an additional
anticancer agent. The present compositions, which comprise a
Synergistic Polyphenol Compound, can be administered orally. The
compositions can also be administered by any other convenient
route, for example, by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral, rectal,
and intestinal mucosa, etc.) and can be administered together with
another biologically active agent. Administration can be systemic
or local. Various delivery systems are known, e.g., encapsulation
in liposomes, microparticles, microcapsules, capsules, etc., and
can be administered.
[0162] Methods of administration include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, oral, sublingual,
intracerebral, intravaginal, transdermal, rectal, by inhalation, or
topical, particularly to the ears, nose, eyes, or skin. In some
instances, administration will result in the release of the
Synergistic Polyphenol Compounds into the bloodstream. The mode of
administration is left to the discretion of the practitioner.
[0163] In one embodiment, the Synergistic Polyphenol Compounds or
Synergistic Polyphenol Compositions are administered orally.
[0164] In another embodiment, the Synergistic Polyphenol Compounds
or Synergistic Polyphenol Compositions are administered
intravenously.
[0165] In other embodiments, it can be desirable to administer the
Synergistic Polyphenol Compounds or Synergistic Polyphenol
Compositions locally. This can be achieved, for example, and not by
way of limitation, by local infusion during surgery, topical
application, e.g., in conjunction with a wound dressing after
surgery, by injection, by means of a catheter, by means of a
suppository or enema, or by means of an implant, said implant being
of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers.
[0166] In certain embodiments, it can be desirable to introduce the
Synergistic Polyphenol Compounds or Synergistic Polyphenol
Compositions into the central nervous system or gastrointestinal
tract by any suitable route, including intraventricular,
intrathecal, and epidural injection, and enema. Intraventricular
injection can be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
[0167] Pulmonary administration can also be employed, e.g., by use
of an inhaler of nebulizer, and formulation with an aerosolizing
agent, or via perfusion in a fluorocarbon or a synthetic pulmonary
surfactant. In certain embodiments, the Synergistic Polyphenol
Compounds or Synergistic Polyphenol Compositions can be formulated
as a suppository, with traditional binders and excipients such as
triglycerides.
[0168] In another embodiment the Synergistic Polyphenol Compounds
or Synergistic Polyphenol Compositions can be delivered in a
vesicle, in particular a liposome (see Langer, Science
249:1527-1533 (1990) and Treat or prevent et al., Liposomes in the
Therapy of Infectious Disease and Cancer 317-327 and 353-365
(1989)).
[0169] In yet another embodiment the Synergistic Polyphenol
Compounds or Synergistic Polyphenol Compositions can be delivered
in a controlled-release system or sustained-release system (see,
e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)). Other controlled or
sustained-release systems discussed in the review by Langer,
Science 249:1527-1533 (1990) can be used. In one embodiment a pump
can be used (Langer, Science 249:1527-1533 (1990); Sefton, CRC
Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980); and Saudek et al., N. Engl. J. Med. 321:574 (1989)).
In another embodiment polymeric materials can be used (see Medical
Applications of Controlled Release (Langer and Wise eds., 1974);
Controlled Drug Bioavailability, Drug Product Design and
Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J.
Macromol. Sci. Rev. Macromol. Chem. 2:61 (1983); Levy et al.,
Science 228:190 (1935); During et al., Ann. Neural. 25:351 (1989);
and Howard et al., J. Neurosurg. 71:105 (1989)).
[0170] In yet another embodiment a controlled- or sustained-release
system can be placed in proximity of a target of the Synergistic
Polyphenol Compounds or Synergistic Polyphenol Compositions, e.g.,
the spinal column, brain, heart, abdomen, thoracic cavity, skin,
lung, or gastrointestinal tract, thus requiring only a fraction of
the systemic dose.
[0171] The present compositions can optionally comprise a suitable
amount of a physiologically acceptable excipient so as to provide
the form for proper administration to the subject.
[0172] Such physiologically acceptable excipients can be liquids,
such as water and oils, including those of petroleum, subject,
vegetable, or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. The physiologically
acceptable excipients can be saline, gum acacia; gelatin, starch
paste, talc, keratin, colloidal silica, urea and the like. In
addition, auxiliary, stabilizing, thickening, lubricating, and
coloring agents can be used. In one embodiment the physiologically
acceptable excipients are sterile when administered to a subject.
Water is a particularly useful excipient when the composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid excipients,
particularly for injectable solutions. Suitable excipients also
include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, sodium stearate, glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene,
glycol, water, ethanol and the like. The present compositions, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents.
[0173] The present compositions can take the form of solutions,
suspensions, emulsions, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, aerosols, sprays, or any other form suitable for
use. In one embodiment the composition is in the form of a capsule
(see e.g. U.S. Pat. No. 5,698,155). Other examples of suitable
physiologically acceptable excipients are described in Remington's
Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th
ed. 1995), incorporated herein by reference.
[0174] In one embodiment the Synergistic Polyphenol Compounds or
Synergistic Polyphenol Compositions are formulated in accordance
with routine procedures as a composition adapted for oral
administration to human beings. Compositions for oral delivery can
be in the form of tablets, lozenges, aqueous or oily suspensions,
granules, powders, emulsions, capsules, syrups, or elixirs for
example. Orally administered compositions can contain one or more
agents, for example, sweetening agents such as fructose, aspartame
or saccharin; flavoring agents such as peppermint, oil of
wintergreen, or cherry; coloring agents; and preserving agents, to
provide a pharmaceutically palatable preparation. Moreover, where
in tablet or pill form, the compositions can be coated to delay
disintegration and absorption in the gastrointestinal tract thereby
providing a sustained action over an extended period of time.
Selectively permeable membranes surrounding an osmotically active
platform driving a Synergistic Polyphenol Composition are also
suitable for orally administered compositions. In these latter
platforms, fluid from the environment surrounding the capsule is
imbibed by the driving compound, which swells to displace the agent
or agent composition through an aperture. These delivery platforms
can provide an essentially zero order delivery profile as opposed
to the spiked profiles of immediate release formulations. A
time-delay material such as glycerol monostearate or glycerol
stearate can also be used. Oral compositions can include standard
excipients such as mannitol, lactose, starch, magnesium stearate,
sodium saccharin, cellulose, and magnesium carbonate. In one
embodiment the excipients are of pharmaceutical grade.
[0175] In another embodiment the compositions can be formulated for
intravenous administration. Typically, compositions for intravenous
administration comprise sterile isotonic aqueous buffer. Where
necessary, the compositions can also include a solubilizing agent.
Compositions for intravenous administration can optionally include
a local anesthetic such as lignocaine to lessen pain at the site of
the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized-powder or water free concentrate in a hermetically
sealed container such as an ampule or sachette indicating the
quantity of active agent. Where the compositions are to be
administered by infusion, they can be dispensed, for example, with
an infusion bottle containing sterile pharmaceutical grade water or
saline. Where the compositions are administered by injection, an
ampule of sterile water for injection or saline can be provided so
that the ingredients can be mixed prior to administration.
[0176] The compositions can be administered by controlled-release
or sustained-release means or by delivery devices that are well
known to one skilled in the art. Examples include, but arc not
limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;
3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;
5,120,548; 5,073,543; 5,639,476; 5,354;556; and 5,733,556, each of
which is incorporated herein by reference. Such dosage forms can be
used to provide controlled- or sustained-release of one or more
active components using, for example, hydropropylmethyl cellulose,
other polymer matrices, gels, permeable membranes, osmotic systems,
multilayer coatings, microparticles, liposomes, microspheres, or a
combination thereof to provide the desired release profile in
varying proportions. Suitable controlled- or sustained-release
formulations known to one skilled in the art, including those
described herein, can be readily selected for use with the active
components of the invention. The invention thus encompasses single
unit dosage forms suitable for oral administration such as, but not
limited to, tablets, capsules, gelcaps, and caplets that are
adapted for controlled- or sustained-release.
[0177] In one embodiment a controlled- or sustained-release
composition comprises a minimal amount of one or more Synergistic
Polyphenol Compounds so as to treat or prevent cancer in a minimal
amount of time. Advantages of controlled- or sustained-release
compositions include extended activity of the drug, reduced dosage
frequency, and increased subject compliance. In addition,
controlled- or sustained-release compositions can favorably affect
the time of onset of action or other characteristics, such as blood
levels of the Synergistic Polyphenol Compounds, and can thus reduce
the occurrence of adverse side effects.
[0178] Controlled- or sustained-release compositions can initially
release an amount of a Synergistic Polyphenol Compound that
promptly produces the desired therapeutic or prophylactic effect,
and gradually and continually release other amounts of the
Synergistic Polyphenol Compounds to maintain this level of
therapeutic or prophylactic effect over an extended period of time.
To maintain a constant level of a Synergistic Polyphenol Compound
in the body, the Synergistic Polyphenol Compound can be released
from the dosage form at a rate that will replace the amount of
Synergistic Polyphenol Compound being metabolized and excreted from
the body. Controlled- or sustained-release of a Synergistic
Polyphenol Composition or a Synergistic Polyphenol Compound
component of a Synergistic Polyphenol Composition can be stimulated
by various conditions, including but not limited to, changes in pH,
changes in temperature, concentration or availability of enzymes,
concentration or availability of water, or other physiological
conditions or compounds.
[0179] In one embodiment, the Synergistic Polyphenol Compositions
comprise an amount of each the Synergistic Polyphenol Compounds
which together are effective to treat cancer. In another
embodiment, the Synergistic Polyphenol Compositions comprise an
amount of the Synergistic Polyphenol Compounds which are effective
to treat cancer when each of the compounds are administered
separately as monotherapy. Typically, the compositions of the
invention comprise at least about 0.01% of the combined Synergistic
Polyphenol Compounds by weight of the composition. When intended
for oral administration, this amount can be varied to be between
0.1% and 80% by weight of the composition. In one embodiment, an
oral composition can comprise from between 4% and 50% of combined
amount of the Synergistic Polyphenol Compounds by weight of the
composition. In another embodiment, compositions of the present
invention are prepared so that a parenteral dosage unit contains
from between 0.01% and 2% by weight of the combined amount of the
Synergistic Polyphenol Compounds of the invention.
[0180] A Synergistic Polyphenol Compound can administered to a
subject at dosages from about 1 mg/m.sup.2 to about 1000
mg/m.sup.2, from about 100 mg/m.sup.2 to about 700 mg/m.sup.2, or
from about 200 mg/m.sup.2 to about 500 mg/m.sup.2. The dosage
administered is dependent upon various parameters, including, but
not limited to, the cancer being treated, the subject's general
health, and the administering physician's discretion. In specific
embodiments, the total combined dosage of all Synergistic
Polyphenol Compounds administered to a subject is about 50
mg/m.sup.2, about 75 mg/m.sup.2, about 100 mg/m.sup.2, about 125
mg/m.sup.2, about 150 mg/m.sup.2, about 175 mg/m.sup.2, about 200
mg/m.sup.2, about 225 mg/m.sup.2, about 250 mg/m.sup.2, about 275
mg/m.sup.2, about 300 mg/m.sup.2, about 325 mg/m.sup.2, about 350
mg/m.sup.2, about 375 mg/m.sup.2, about 400 mg/m.sup.2, about 425
mg/m.sup.2, about 450 mg/m.sup.2, about 475 mg/m.sup.2, about 500
mg/m.sup.2, about 525 mg/m.sup.2, about 550 mg/m.sup.2, about 575
mg/m.sup.2, about 600 mg/m.sup.2, about 625 mg/m.sup.2, about 650
mg/m.sup.2, about 675 mg/m.sup.2, about 700 mg/m.sup.2, about 725
mg/m.sup.2, about 750 mg/m.sup.2, about 775 mg/m.sup.2, about 800
mg/m.sup.2, about 825 mg/m.sup.2, about 850 mg/m.sup.2, about 875
mg/m.sup.2, about 900 mg/m.sup.2, about 925 mg/m.sup.2, about 950
mg/m.sup.2, about 975 mg/m.sup.2, or about 1000 mg/m.sup.2.
[0181] The amount of the Synergistic Polyphenol Compounds that is
effective in the treatment or prevention of cancer can be
determined by standard clinical techniques. In addition, in vitro
or in vivo assays can optionally be employed to help identify
optimal dosage ranges. The precise dose to be employed will also
depend on the identity of the Synergistic Polyphenol Compounds
being administered, route of administration, and the seriousness of
the condition being treated and should be decided according to the
judgment of the practitioner and each subject's circumstances in
view of, e.g., published clinical studies. Suitable effective
amounts for each Synergistic Polyphenol Compound being
administered, however, range from about 10 micrograms to about 5
grams. In certain embodiments, the effective amount is about 50 mg,
about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500
mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1
g, about 1.2 g, about 1.4 g, about 1.6 g, about 1.8 g, about 2.0 g,
about 2.2 g, about 2.4 g, about 2.6 g, about 2.8 g, and about 3.0
g. Dosages may be administered over various time periods including,
but not limited to, about every 2 hours, about every 6 hours, about
every 8 hours, about every 12 hours, about every 24 hours, about
every 36 hours, about every 48 hours, about every 72 hours, about
every week, about every two weeks, about every three weeks, about
every month, and about every two months.
[0182] In one embodiment, the Synergistic Polyphenol Compounds are
administered concurrently to a subject in separate compositions.
The Synergistic Polyphenol Compounds may be administered to a
subject by the same or different routes of administration.
[0183] When the Synergistic Polyphenol Compounds are administered
to a subject concurrently, the term "concurrently" is not limited
to the administration of the Synergistic Polyphenol Compounds at
exactly the same time, but rather it is meant that they are
administered to a subject in a sequence and within a time interval
such that they can act synergistically to provide an increased
benefit than if they were administered otherwise. For example, the
Synergistic Polyphenol Compounds may be administered at the same
time or sequentially in any order at different points in time;
however, if not administered at the same time, they should be
administered sufficiently close in time so as to provide the
desired therapeutic effect, preferably in a synergistic fashion.
The Synergistic Polyphenol Compounds can be administered
separately, in any appropriate form and by any suitable route. When
the Synergistic Polyphenol Compounds are not administered in the
same composition, it is understood that they can be administered in
any order to a subject in need thereof. For example, a first
Synergistic Polyphenol Compound can be administered prior to (e.g.,
5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks after) the administration of a second Synergistic Polyphenol
Compound, to a subject in need thereof. In various embodiments the
Synergistic Polyphenol Compounds are administered concurrently, 1
minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour
apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours
apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours
to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours
apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10
hours to 11 hours apart, 11 hours to 12 hours apart, no more than
24 hours apart or no more than 48 hours apart. In one embodiment,
the Synergistic Polyphenol Compounds are administered within the
same office visit. In another embodiment, the Synergistic
Polyphenol Compounds are administered at 1 minute to 24 hours
apart.
[0184] Suitable effective dosage amounts for the Synergistic
Polyphenol Compositions are based upon the total amount of the
Synergistic Polyphenol Compounds present in the composition. For
the Synergistic Polyphenol Compositions of the present, the total
amount of Synergistic Polyphenol Compounds should be within a range
of from about 0.01 to about 100 w/w. The effective dosage amounts
described herein refer to the total amounts of all Synergistic
Polyphenol Compounds administered. If one or more Synergistic
Polyphenol Composition is administered, the effective dosage
amounts correspond to the combined amount of all Synergistic
Polyphenol Compounds in each of the Synergistic Polyphenol
Compositions administered.
[0185] The Synergistic Polyphenol Compounds or Synergistic
Polyphenol Compositions can be assayed in vitro or in vivo for the
desired therapeutic or prophylactic activity prior to use in
humans. Subject model systems can be used to demonstrate safety and
efficacy.
[0186] The present methods for treating or preventing cancer in a
subject can further comprise administering another therapeutic
agent to the subject being administered a Synergistic Polyphenol
Compound or Synergistic Polyphenol Composition. In one embodiment
the other therapeutic agent is administered in an effective
amount.
[0187] Effective amounts of the other therapeutic agents are well
known to one skilled in the art. However, it is well within the
skilled artisan's purview to determine the other therapeutic
agent's optimal effective amount range.
[0188] In one embodiment, the other therapeutic agent is an
antiemetic agent.
[0189] In yet another embodiment the other therapeutic agent is a
hematopoietic colony stimulating factor.
[0190] In a further embodiment, the other therapeutic agent is an
agent useful for reducing any potential side effect of a
Synergistic Polyphenol Composition, a Synergistic Polyphenol
Compound, or another anticancer agent.
[0191] In one embodiment, the Synergistic Polyphenol Compounds or
Synergistic Polyphenol Compositions can be administered prior to,
at the same time as, or after an antiemetic agent, or on the same
day, or within 1 hour, 2 hours, 12 hours, 24 hours, 48 hours or 72
hours of each other.
[0192] In another embodiment, the Synergistic Polyphenol Compounds
or Synergistic Polyphenol Compositions can be administered prior
to, at the same time as, or after a hematopoietic
colony-stimulating factor, or on the same day, or within 1 hour, 2
hours, 12 hours, 24 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3
weeks or 4 weeks of each other.
[0193] The Synergistic Polyphenol Compounds or Synergistic
Polyphenol Compositions of the present invention can also be
cyclically administered. Cycling therapy involves the
administration of one Synergistic Polyphenol Compound or
Synergistic Polyphenol Composition for a period of time, followed
by the administration of a second Synergistic Polyphenol Compound
or Synergistic Polyphenol Composition for a period of time and
repeating this sequential administration, i.e., the cycle, in order
to reduce the development of resistance to one or both of the
Synergistic Polyphenol Compounds or Synergistic Polyphenol
Compositions, to avoid or reduce the side effects of one or both of
the Synergistic Polyphenol Compounds or Synergistic Polyphenol
Compositions, and/or to improve the efficacy of the treatment.
[0194] In one embodiment, the cycling therapy includes one or more
other anticancer agents in addition to the Synergistic Polyphenol
Compounds or Synergistic Polyphenol Compositions.
[0195] In one embodiment, the Synergistic Polyphenol Compounds or
Synergistic Polyphenol Compositions are administered concurrently
to a subject in separate compositions. The Synergistic Polyphenol
Compounds or Synergistic Polyphenol Compositions may be
administered to a subject by the same or different routes of
administration.
5.6 Kits
[0196] The invention encompasses kits that can simplify the
administration of a Synergistic Polyphenol Compound or a
Synergistic Polyphenol Composition to a subject.
[0197] A typical kit of the invention comprises a unit dosage form
of a Synergistic Polyphenol Compound or a Synergistic Polyphenol
Composition. In one embodiment the unit dosage form is a container,
which can be sterile, containing an effective amount of a
Synergistic Polyphenol Compound or a Synergistic Polyphenol
Composition, and a physiologically acceptable carrier or vehicle.
The kit can further comprise a label or printed instructions
instructing the use of the Synergistic Polyphenol Compound or a
Synergistic Polyphenol Composition to treat or prevent cancer in a
subject. The kit can also further comprise a unit dosage form of
another therapeutic agent, for example, a container containing an
effective amount of the other therapeutic agent. In one embodiment
the kit comprises a container containing an effective amount of a
Synergistic Polyphenol Compound or a Synergistic Polyphenol
Composition and an effective amount of another therapeutic agent.
Examples of other therapeutic agents include, but are not limited
to, those listed above.
[0198] Kits of the invention can further comprise a device that is
useful for administering the unit dosage forms. Examples of such a
device include, but are not limited to, a syringe, a drip bag, a
patch, an inhaler, and an enema bag.
[0199] The following examples are set forth to assist in
understanding the invention and should not, of course, be construed
as specifically limiting the invention described and claimed
herein. Such variations of the invention, including the
substitution of all equivalents now known or later developed, which
would be within the purview of one skilled in the art, and changes
in formulation or minor changes in experimental design, are to be
considered to fall within the scope of the invention incorporated
herein.
6. EXAMPLES
[0200] Chemicals. (-)-epigallocatechin gallate and Poly E were
provided by the National Cancer Institute (Bethesda, Md.). Poly E
contains about 60% (-)-epigallocatechin gallate, 7%
(-)-epicatechin, 12% EGC, 1% (-)-epicatechin gallate, and 2%
(-)-gallocatechin gallate, respectively.
[0201] Cell lines and cell culture. The Caco2, HCT116, HT29, SW480,
and SW837 human colorectal cancer cell lines and the FHC normal
human fetal colon cell line were obtained from American Type
Culture Collection (Manassas, Va.). All cell lines were maintained
in DF10 medium containing DMEM (Invitrogen, San Diego, Calif.)
supplemented with 10% fetal bovine serum (Invitrogen). The FHC cell
line was originally established from the colon of a human fetus at
13 weeks of gestation and has an epithelial morphology. Cells were
cultured in an incubator with humidified air at 37.degree. C. with
5% CO.sub.2. As an untreated solvent control, cells were treated
with DMSO (Sigma Chemical Co., St. Louis, Mo.) at a final
concentration of <0.1%.
6.1 Example 1
Extraction of Polyphenols From Green Leaves
[0202] Crude polyphenols can be extracted from Chinese green tea
leaves (500 g) using hot water, as described in European Patent No.
EP 1402869 to Schneider, then dissolved in ethanol (5 mL). The
ethanolic solution is then loaded onto a C16/100 chromatographic
column (1.6 cm.times.90 cm, Sephadex LH-20, equilibrated using
ethanol) and chromatographed using a flow rate of about 1.2 mL/min.
Collected fractions can be first analyzed using thin-layer
chromatography on silica gel plates (chloroform/methanol/water
(65:35:10, v/v/v as eluent) and subsequently developing the eluted
plates using a spray reagent that is prepared by dissolving 1 g
vanillin in 50 mL concentrated HCl. Fractions that are positive to
the spray reagent can be concentrated in vacuo, and the resulting
residue dissolved in methanol. The resulting methanolic solution
can be analyzed using UV/visible spectroscopy by measuring their
absorbances at 280 nm and 500 nm for detecting the presence of
polyphenols. Fractions containing crude polyphenols can be purified
using the methods described in Hoefler et al., J. Chromatogr.
129:460-3 (1976).
6.2 Example 2
Cell Viability Assay
[0203] Cell viability assays were performed as described in Suzui
et al., Cancer Res. 2002; 62:3997-4006, using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
cell proliferation kit I (Roche Diagnostics Co., Indianapolis,
Ind.), according to the instructions of the manufacturer. Cells to
be tested against were plated onto 96-well plates (3.times.10.sup.3
cells/well) and twenty-four hours later, the cells were treated
with an illustrative Synergistic Polyphenol Composition, or one or
more illustrative Synergistic Polyphenol Compounds. Neither the
medium nor the test compounds were changed during this time period.
The assay was done in triplicate.
6.3 Example 3
Determination of the Synergistic Anticancer Properties of Selected
Synergistic Polyphenol Compounds Against a Human Colorectal Cancer
Cell Line
[0204] Three thousand HT29 cells were plated onto 96-well plates.
Twenty-four hours later, (-)-epigallocatechin gallate and
(-)-epicatechin were added alone or in combination at the
concentrations indicated above in Example 2 to each well, and the
cells were incubated for 48 hours in DF10 medium. cell viability
assays were then done using the MTT system as described in Example
2. To determine whether the combined effects of
(-)-epigallocatechin gallate plus (-)-epicatechin were synergistic,
the combination index isobologram was used to analyze the data
obtained as described, for example, in Soriano et al., Cancer Res.
1999; 59:6178-84 and Shimizu et al., Clin. Cancer Res.
2004;10:6710-2. An isobologram corresponding to the collected data
is shown in FIG. 3, while Table 3 summarizes the results of the
isobologram analysis. TABLE-US-00003 TABLE 3 Summary of the
Isobologram Analysis of the Effect of Combinations of
(-)-epigallocatechin gallate and (-)-epicatechin on the Viability
of a Human Colorectal Cancer Cell Line (-)-epicatechin
(-)-epigallocatechin gallate concentration concentration (.mu.g/mL)
(.mu.g/mL) 0.1 1 10 20 1 - - + ++ 10 - .+-. + ++ 50 .+-. .+-. ++
+++ 100 .+-. .+-. ++ +++ - indicates no synergy .+-. indicates a
moderate additive effect + indicates slight synergism ++ indicates
moderate synergism +++ indicates synergism
[0205] These results indicate that a combination of the Synergistic
Polyphenol Compounds (-)-epigallocatechin gallate and
(-)-epicatechin causes synergistic inhibitions of growth in a human
cancer cell line. Accordingly, the Synergistic Polyphenol Compounds
or Synergistic Polyphenol Compositions are useful for treating
colorectal cancer.
6.4 Example 4
Determination of the Anti-Proliferative Effects of Illustrative
Synergistic Polyphenol Compounds in a Human Colorectal Cancer Cell
Line
[0206] One hundred thousand HT29 cells were seeded in multiple
35-mm diameter dishes in DF10 medium, and 24 hours later they were
treated with (-)-epigallocatechin gallate or Poly E for 96 hours at
a concentration of 1 or 20 .mu.g/mL. The medium and test compounds
were not changed during this time period. As a control, the cells
were treated with 0.1% DMSO. Every 24 hours up to 96 hours,
replicate plates of cells were washed with phosphate-buffered
saline and the attached cells were collected using trypsinization.
The numbers of cells in replica plates were then determined using a
Coulter Counter (Beckman Coulter Co., Fullerton, Calif.) as
described in Shimizu et al., Clin. Cancer Res. 2004: 10:1130-40.
Table 4 shows IC.sub.50 values for both (-)-epigallocatechin
gallate and Poly E against various human colorectal cancer cell
lines. TABLE-US-00004 TABLE 4 IC50 values for (-)-epigallocatechin
gallate and Poly E against human colorectal cancer cell lines
IC.sub.50 (-)- (-)- epigallocatechin epigallocatechin Cell gallate
gallate Poly E Poly E Line (.mu.g/mL) (.mu.mol/L) (.mu.g/mL)
(.mu.mol/L) Caco2 19.2 42.2 22.3 29.4 HCT116 21.7 47.7 20.6 27.2
HT29 22.8 50.2 20.1 26.5 SW480 36.4 80.1 37.0 48.4 SW837 19.6 43.1
19.6 25.9 FHC 48.8 107.4 49.4 65.2
[0207] These results suggest that ECGC and Poly E preferentially
inhibit the growth of human colorectal cancer cells and
accordingly, the Synergistic Polyphenol Compounds or Synergistic
Polyphenol Compositions are useful for treating colorectal
cancer.
6.5 Example 5
Induction of Apoptosis in a Human Colorectal Cancer Cell Line
[0208] To quantify the induction of apoptosis in human colorectal
cancer cell lines, a DNA fragmentation assay can be performed using
the Cell Death Detection ELISA.sup.PLUS kit (Roche Diagnostics), as
described in Shimizu et al., Clin. Cancer Res. 2004; 10:6710-2, and
Shimizu et al., Clin. Cancer Res. 2004: 10:1130-40. In this assay,
human colorectal cancer cell cells can be seeded into 6-cm diameter
dishes (5.times.10.sup.5 cells/dish) in DF10 medium, and 24 hours
later the cells are treated with a test compound for an appropriate
time as set forth in the cited references.
6.6 Example 6
Protein Extraction, Immunoprecipitation, and Western Blot
Analysis
[0209] Total cellular protein was extracted and equivalent amounts
of protein can be determined by western blot analysis using the
methods described in Suzui, et al., Cancer Res., 62:3997-4006
(2002). The primary antibodies for several proteins are described
in Masuda et al., Clin Cancer Res., 7:4220-4229 (2001); Masuda et
al., Clin Cancer Res., 9:3486-3491 (2003); and Shimizu et al., Clin
Cancer Res., 11:2735-2746 (2005). The primary antibodies for HER3
(2F12) and COX-2 (PG27) were purchased from Upstate Cell Signaling
Solutions (Charlottesville, Va.) and Oxford Biochemical Research
(Oxford, Mich.), respectively. To examine the levels of expression
of the phosphorylated (i.e. activated) form of the HER3 protein
(p-HER3), cellular lysates (1 mg/mL) were incubated with 4 .mu.g
anti-HER3 antibody overnight at 4.degree. C. with slow agitation,
and immune complexes were then precipitated by adding protein A/G
agarose (sc-2003, Santa Cruz Biotechnology, Santa Cruz, Calif.).
After incubating for 2 hours at 4.degree. C., the beads were washed
with lysis buffer, and bound proteins were eluted into the sample
buffer and subjected to western blot analysis, as described above.
The p-HER3 protein was then detected by using an
anti-phospho-tyrosine antibody (PY-20-HRP, BD Transduction
Laboratories, San Diego, Calif.). An antibody to actin was used as
a loading control. Each membrane was developed using an
ECL-enhanced chemiluminescence system (Amersham Biosciences,
Piscataway, N.J.).
6.7 Example 7
RNA Extraction and Semiquantitative RT-PCR Analysis
[0210] RNA extraction and semiquantitative RT-PCR analysis were
performed using the methods described in Suzui, et al., Cancer
Res., 62:3997-4006 (2002). Total RNA was isolated from SW837 human
colon cancer cells using Trizol reagent (Invitrogen) according to
the manufacturer's instructions. cDNA was amplified from 1 .mu.g of
total RNA using SuperScript one-step RT-PCR with the platinum Taq
system (Invitrogen). PCR was conducted for 25 cycles in a thermal
controller (Programmable Thermal Controller; MJ Research Inc.,
Watertown, Mass.). The COX-2 specific primer sets F1COX-2 (5'-GTT
CCC ACC CAT GTC AAA AC-3') and R2COX-2 (5'-GCC CTC GCT TAT GAT CTG
TC-3') were designed using the sequence described in Hla et al.,
Proc. Natl. Acad. Sci. USA, 89:7384-7388 (2004). Actin specific
primer sets and the amplification cycle conditions were as
described in Suzui, et al., Cancer Res., 62:3997-4006 (2002). The
intensities of mRNA bands were quantified using NIH Image software
version 1.62.
6.8 Example 8
DNA Fragmentation Assays
[0211] To quantify the induction of apoptosis, a DNA fragmentation
assay was performed using the Cell Death Detection ELISA.sup.PLUS
kit (Roche Diagnostics Co., Indianapolis, Ind.), according to the
manufacturer's instructions and as described in Shimizu et al.,
Clin. Cancer Res., 10:1130-1140 (2004). SW837 human colon cancer
cells (5.0.times.10.sup.5 cells/6-cm diameter dish) were treated
with EGCG (1.0 or 20 .mu.g/mL) for 48 or 96 hours, and samples were
then prepared. Control cells were treated only with DMSO. The
extent of DNA fragmentation at time 0 hours was set to 1 and
increases expressed as fold activation.
6.9 Example 9
COX-2, AP-1, and NF-.kappa.B Reporter Assays
[0212] Reporter assays were performed performed using the methods
described in Suzui, et al., Cancer Res., 62:3997-4006 (2002). The
COX-2-luciferase reporter plasmid phPES2(-327/+59) was provided by
the National Cardiovascular Research Institute (Osaka, Japan). The
pAP-1 luciferase reporter plasmid was provided by the National
Cancer Institute (Bethesda, Md.). The NF-.quadrature.B promoter
luciferase reporter plasmid .quadrature.NF-.quadrature.B-Luc is
described in Masuda et al., Clin. Cancer Res. 7:4220-4229 (2001).
One .mu.g of DNA of the indicated luciferase reporter plasmid was
transfected into SW837 human colon cancer cells (3.0.times.10.sup.5
cells/35-mm diameter dish) using Lipofectin reagent (Invitrogen) in
opti-MEM I medium (Invitrogen). After 24 hours the cells were
treated with EGCG (0, 10, or 20 .mu.g/mL) for 24 hours, and
extracts were then prepared. Luciferase activities were determined
using a luciferase assay system (Program Co., Madison, Wis.). In
all of these reporter assays the cells were also cotransfected with
a CMV-.quadrature.-galactosidase (.quadrature.-gal) reporter.
Luciferase activities were normalized with respect to the
.quadrature.-gal activities, to correct for differences in
transfection efficiency, using the methods described in Suzui, et
al., Cancer Res., 62:3997-4006 (2002).
6.10 Example 10
PGE.sub.2 Production Assays
[0213] SW837 human colon cancer cells were plated into 35-mm dishes
and grown to 60% confluence. The cells were then treated with EGCG
(0, 10, 20, or 40 .mu.g/mL) in serum minus medium containing 20
.mu.M arachidonic acid (Sigma) for 18 hours. The cell free medium
was then collected and the amounts of PGE.sub.2 released by cells
into the medium were measured using an EIA kit (Cayman Chemicals,
Ann Arbor, Mich.), according to the manufacturer's instructions.
Production of PGE.sub.2 was normalized to the protein concentration
of the cells.
6.11 Example 11
Cell Proliferation Assays
[0214] Thirty thousand SW837 human colon cancer cells were seeded
into multiple 35-mm diameter dishes in DFI0 medium, and 24 hours
later the cells were treated with EGCG (0, 1.0, or 20 .mu.g/mL) for
96 hours, during which time, the medium and drugs were not changed.
As a control, the cells were treated with 0.1% DMSO. Every 24
hours, and for a total time of 96 hours, replicate plates of cells
were washed with PBS and the attached cells were collected by
trypsinization. The numbers of cells in replica plates were then
counted using a Coulter Counter (Beckman Coulter Co., Fullerton,
Calif.) as described in Shimizu et al., Clin. Cancer Res.,
10:1130-1140 (2004).
6.12 Example 12
Statistical Analysis
[0215] Statistical analyses of DNA fragmentation assays, reporter
assays, and PGE.sub.2 production assays were analyzed by Student's
t test. The results were considered statistically significant if
the P value was less than 0.05.
6.13 Example 13
The COX-2 and HER3 Proteins are Overexpressed in SW837 and Caco2
Colon Cancer Cells
[0216] Using western blot analysis as described above in Example 6,
it was determined that the level of the COX-2 protein was markedly
increased in SW837 human colon cancer cells and was expressed at a
moderate level in Caco2 cells, as shown in FIG. 8(a). The HER3
protein was also expressed at a high level in the SW837 and Caco2.
The level of the p-HER3 protein was also increased in these two
cell lines, indicating constitutive activation of this receptor, as
shown in FIG. 8(b). It has been reported that the EGFR and HER2
proteins are also overexpressed and constitutively activated in
SW837 human colon cancer cells, and that these cells display strong
activation for EGFR, HER2, and HER3. Because activation of members
of the EGF receptor family, especially the HER2/HER3 heterodimer,
has been implicated in the induction of COX-2 expression in colon
cancer cells, SW837 human colon cancer cells are thereby useful in
studies involving HER2 and COX-2.
6.14 Example 14
EGCG Inhibits Activation of EGFR, HER2, and HER3 Signaling
Pathways, Inhibits Expression of COX-2, and Induces Apoptosis in
SW837 human colon cancer cells
[0217] It is well-established that abnormalities in the EGFR family
of RTKs play critical roles in the development of various types of
cancer, and it is reported that the EGFR and HER2 receptors are
targets of EGCG, an illustrative Synergistic Polyphenol Compound1,
in a variety of cancer cell lines. It was examined in parallel
whether EGCG, an illustrative Synergistic Polyphenol Compound,
inhibits activation of EGFR, HER2, and HER3, and related downstream
signaling pathways in SW837 colon cancer cells. Also examined was
the effect of EGCG on cellular levels of both COX-2 protein (FIG.
9(a)) and mRNA (FIG. 9(b)) in SW837 human colon cancer cells. Time
course studies indicate that when the cells were treated with 20
.mu.g/mL EGCG, the IC.sub.50 concentration determined by MTT assays
as described in Shimizu et al., Clin. Cancer Res., 11:2735-2746
(2005), there was a marked decrease in the levels of the p-EGFR,
p-HER2, and p-HER3 proteins within 6 hours after the addition of
EGCG. The levels of the p-Akt and p-ERK proteins displayed marked
decreases at 6 and 12 hours, respectively, after the addition of
EGCG. During this period of time there were no significant changes
in the total levels of the respective proteins, indicating that
EGCG specifically inhibited activation of EGFR, HER2, and HER3; and
also activation of the downstream signaling molecules Akt and ERK.
Results indicate that the cellular level of the COX-2 protein
displayed a marked decrease after 12 to 24 hours, and was almost
undetectable at 48 hours, after treatment with EGCG, as shown in
FIG. 9(a). Semiquantitative RT-PCR studies indicated that EGCG
caused a progressive decrease in the cellular level of the COX-2
mRNA beginning at about 6 hours, with a marked decrease at 48
hours, as shown in FIG. 9(b). In addition, DNA fragmentation
assays, performed as described in Example 8 above, indicate that
treatment with EGCG for 48 hours caused induction of apoptosis in
SW837 human colon cancer cells, as shown in FIG. 9(c). Accordingly,
the Synergistic Polyphenol Compounds are useful for treating human
colorectal cancer.
6.15 Example 15
EGCG Inhibits Transcriptional Activity of the COX-2, AP-1, and
NF-.kappa.B Promoters
[0218] There is evidence that the transcription factor AP-1, which
lies downstream of ERK, plays an important role in activating
expression of COX-2 via the CRE element in the COX-2 promoter.
Increased binding of AP-1 to CRE in the COX-2 promoter occurs in
HER2 transformed human mammary epithelial cells. There is also
evidence that the NF-.quadrature.B signaling pathway, which is
activated by the Akt pathway, plays an important role in regulating
COX-2 expression via the NF-.quadrature.B binding site in the COX-2
promoter. The effects of EGCG, an illustrative Synergistic
Polyphenol Compound, on the transcriptional activity of the COX-2,
AP-1, and NF-.quadrature.B promoters, was determined using
transient transfection luciferase reporter assays. As shown in FIG.
10, exposure of SW837 human colon cancer cells to EGCG (0, 10 and
20 .mu.g/mL)caused inhibition of COX-2 (FIG. 10(a)), AP-1(B) (FIG.
10(b)), and NF-.quadrature.B (FIG. 10(c)) luciferase reporter
activities. EGCG induced inhibition was most apparent with respect
to the AP-1 promoter activity. Taken together with the results of
the study described in Example 14 above, which indicates that EGCG
causes a decrease in cellular levels of both COX-2 protein and
mRNA, these results provide evidence that the inhibitory effects of
EGCG on expression of COX-2 are exerted, at least in part, at the
level of gene transcription, presumably through inhibition of
activation of the EGFR, HER2, and HER3 receptors, and downstream
signaling molecules, such as ERK and Akt.
6.16 Example 16
EGCG Causes a Decrease in the Production of PGE.sub.2 by SW837
human colon cancer cells
[0219] Treatment of SW837 human colon cancer cells with EGCG, an
illustrative Synergistic Polyphenol Compound (0, 10, 20 or 40
.mu.g/mL), resulted in a decrease in the levels of PGE.sub.2, a
major product of the COX-2 enzyme, as indicated in FIG. 11. Because
PGE.sub.2 can enhance the proliferation, migration, motility, and
invasion of colon cancer cells, inhibition of the production of
PGE.sub.2 by EGCG may play a critical role in the ability of this
compound to inhibit growth and induce apoptosis in colon cancer
cells. Accordingly, the Synergistic Polyphenol Compounds are useful
for the treatment of human colorectal cancer.
6.17 Example 17
Longer Exposure to a Low Concentration of EGCG Inhibits Cell
Growth, Induces Apoptosis, Inhibits Activation of the EGFR, HER2,
and HER3 Receptors, and Inhibits Expression of COX-2 and
Bcl-x.sub.L Proteins in SW837 human colon cancer cells
[0220] Using the method described above in Example 11, SW837 human
colon cancer cells were treated with 1.0 .mu.g/mL of EGCG, an
illustrative Synergistic Polyphenol Compound, for 96 hours. This
resulted in an inhibition of growth of about 55%, a significant
increase in a decrease in the levels of the p-EGFR, p-HER2, p-HER3,
COX-2, and Bcl-x.sub.L proteins as indicated in FIG. 12(a), and a
significant increase in apoptosis, as shown in FIG. 12(b).
Accordingly, administration of a low dose of EGCG may inhibit
activation of these RTKs, the expression of COX-2, and the growth
of human colon cancer cells. As such, the Synergistic Polyphenol
Compounds are useful for the treatment of human colorectal
cancer.
6.18 Example 18
Protein Extraction and Western Blot Analysis for IGF
[0221] Total cellular protein was extracted and equivalent amounts
of protein were examined by western blot analysis, as described
above in Example 6. Cell lysates were separated by SDS-PAGE using
7.5 to 15% polyacrylamide gels and transferred onto Immobilon-P
transfer membranes (Millipore Co., Bedford, Mass.). The primary
antibodies for IGF-1R.quadrature.(#3022) and p-IGF-1R (#3021) were
purchased from Cell Signaling Technology (Beverly, Mass.). The
primary antibodies for IGF-1R.quadrature.(sc-713) and IGFBP-3
(sc-6003) were purchased from Santa Cruz Biotechnology (Santa Cruz,
Calif.). The primary antibodies for IGF-1 (clone Sm1.2) and actin
(A2066) were purchased from Upstate Cell Signaling Solutions
(Charlottesville, Va.) and Sigma Chemical Co. (St Louis, Mo.),
respectively. An antibody to actin was used as a loading control.
Anti-mouse IgG (Amersham Pharmacia Biosciences, Piscataway, N.J.),
anti-rabbit IgG (Amersham Pharmacia Biosciences), or anti-goat IgG
(Santa Cruz Biotechnology) antibodies were used as the secondary
antibodies. Each membrane was developed using an ECL-enhanced
chemiluminescence system (Amersham Biosciences).
6.19 Example 19
RNA Extraction and Semiquantitative RT-PCR Analysis
[0222] RNA extraction and semiquantitative RT-PCR analysis were
performed as described above in Example 7. Total RNA was isolated
from SW837 human colon cancer cells using the Trizol reagent
(Invitrogen) according to the manufacturer's instructions. cDNA was
amplified from 1 .mu.g of total RNA using Superscript one-step
RT-PCR with the platinum Taq system (Invitrogen). The primers used
for amplification of IGFBP-3, IGF-1, MMP-7, MMP-9, and
TGF-.quadrature.2 specific genes are shown in FIG. 4. Actin
specific primer sets are described in Suzui, et al., Cancer Res.,
62:3997-4006 (2002). By using a thermal controller (Programmable
Thermal Controller; MJ Research Inc., Watertown, Mass.), 25-, 35-,
32-, 40-, and 30-cycle rounds of PCR were chosen for optimum
analysis of expression of IGFBP-3, IGF-1, MMP-7, MMP-9, and
TGF-.quadrature.2 mRNAs, respectively, since a semiquantitative
assessment indicated that the reactions had not reached a plateau
and were still in the log phase. Each amplification cycle consisted
of 0.5 minutes at 94.degree. C. for denaturation, 0.5 minutes at
55.degree. C. for primer annealing, and 1 minute at 72.degree. C.
for extension. After PCR amplification, the fragments were analyzed
by agarose gel electrophoresis and stained with ethidium bromide.
The intensities of bands were quantified with NIH Image software
version 1.62.
6.20 Example 20
Expression of IGF-1R, p-IGF-1R, IGF-1, and IGFBP-3 Proteins in
Colon Cancer Cell Lines
[0223] Western blot analysis was performed to determine the
cellular levels of the IGF-1R, p-IGF-1R, IGF-1, and IGFBP-3
proteins in five human colon cancer cell lines (HCT116, Caco2,
HT29, SW837 and SW480). Results indicate that the levels of the
IGF-1R.quadrature.and IGF-1R.quadrature.proteins are increased in
the Caco2, HT29, SW837, and SW480 cell lines, especially in SW837
and SW480 cells. The slower migrating pro-IGF-1R protein was also
increased in the SW837 and SW480 cell lines. The level of the
p-IGF-1R protein was also increased in the SW837 and SW480 cell
lines, indicating constitutive activation of this receptor. All
cell lines expressed the IGFBP-3 and IGF-I proteins at
approximately similar levels, as illustrated in FIG. 5.
6.21 Example 21
Effects of EGCG on Expression of IGF-1R and Related Proteins in
SW837 Human Colon Cancer Cells
[0224] Abnormalities in the IGF-1/IGF-1R system play a role an
important role in the development of various types of cancer. Since
EGCG can inhibit activation of several RTKs including EGFR, HER2,
and PDGF, the possible effects of this compound on activation of
IGF-1R and on related proteins in SW837 colon cancer cells was
determined. Time course studies indicated that when the cells were
treated with 20 .mu.g/mL EGCG, the IC.sub.50 concentration
determined using MTT assay, there was a marked decrease in the
levels of the p-IGF-1R protein within 6 hours after the addition of
EGCG, as shown in FIG. 6A. The levels of the IGF-1 protein
displayed a moderate decrease at 12 hours and a marked decrease at
24 hours after the addition of EGCG. Thus, these decreases were
delayed compared with the decrease in the levels of the p-IGF-1R
protein. There was a marked increase in the level of the IGFBP-3
protein at 6 hours after the addition of this compound, and this
increase persisted for at least 48 hours. During this period of
time there were no significant changes in the levels of the
IGF-1R.quadrature.and IGF-1R.quadrature.proteins, indicating that
EGCG specifically inhibited activation of IGF-1R, but did not
inhibit the total amount of these proteins as indicated in FIG.
6A.
[0225] It was then determined whether exposure to a low
concentration (1.0 .mu.g/mL) of EGCG, which is in the range of the
peak plasma concentration of EGCG obtained in a clinical study, for
a longer time period can also alter the levels of p-IGF-1R, IGF-1,
and IGFBP-3 proteins in SW837 human colon cancer cells, as
indicated in FIG. 6B. Results indicate that when the cells were
treated with 1.0 .mu.g/mL of EGCG for 96 hours, there was a
decrease in the levels of the p-EGFR and IGF-1 proteins and an
increase in the IGFBP-3 protein (FIG. 6B), thus reproducing the
effects obtained EGCG at 20 .mu.g/mL, as shown in FIG. 6A.
6.22 Example 22
Effects of EGCG on Expression of IGFBP-3 and IGF-1 mRNAs in SW837
Colon Cancer Cells
[0226] To determine whether the specific changes in cellular levels
of the IGF-1 and IGFBP-3 proteins induced by EGCG shown in FIG. 9A
might be secondary to effects on expression of the corresponding
genes, the effects of EGCG on the levels of IGFBP-3 and IGF-1 mRNAs
were examined using semiquantitative RT-PCR analysis. Time course
studies indicated that treatment with 20 .mu.g/mL EGCG resulted an
increase in the levels of IGFBP-3 mRNA at 3 to 12 hours after the
addition of this compound, and the maximum level peaked at 6 hours.
Results indicate that there is a decrease in the level of IGF-1
mRNA at 12 hours and an additional decline at 24 and 48 hours, as
shown in FIG. 6C.
6.23 Example 23
Effects of EGCG on Expression of MMPs-7 and -9 mRNAs in SW837 Colon
Cancer Cells
[0227] The time-dependent effects of EGCG on expression of MMP-7
and MMP-9 mRNA was measured by treating SW837 human colon cancer
cells with 20 .mu.g/mL EGCG. Results show that there was a marked
decrease in expression of the cellular levels of both MMP-7and
MMP-9 mRNA at 12 hours, and an additional decrease at 24 to 48
hours, as shown in FIG. 7A.
6.24 Example 24
Effects of EGCG on Expression of TGF-.beta.2 mRNA in SW837 Colon
Cancer Cells
[0228] Is is indicated that inhibition of the growth of human
breast cancer cells by TGF.quadrature.2 is associated with
induction of increased levels of IGFBP-3 mRNA and protein, as set
forth in Example 23 and FIG. 7. Therefore, as a possible mechanism
by which EGCG caused an increase in the levels of both IGFBP-3
protein and mRNA in SW837 colon cancer cells, the effects of EGCG
on cellular levels of TGF-.quadrature.2 mRNA were examined using
semiquantitative RT-PCR analysis. Results indicate that the level
of TGF-.quadrature.2 mRNA was transiently increased at 3 hours
after treatment with 20 .mu.g/mL of EGCG, as shown in FIG. 7B.
6.25 Example 25
Effects of EGCG on the Production of IGF-1, IGF-2 and IGFBP-3 in
HepG2 Human Liver Cancer Cells
[0229] Using the MTT cell proliferation assay described in Example
21, and substituting HepG2 human liver cancer cells in place of
SW837 cells, the effects of EGCG (20 .mu.g/mL), an illustrative
Synergistic Polyphenol Compound, on the production of IGF-1, IGF-2
and IGFBP-3 was determined. Results indicate that EGCG at 20
.mu.g/mL inhibited the growth of HepG2 cells with an IC.sub.50 of
21 .mu.g/mL, similar to results obtained with colon cancer cells.
The treatment of HepG2 cells with EGCG also inhibited production of
the growth factors IGF-1 and IGF-2, but increased the production of
IGFBP-3, as shown in FIG. 13. Accordingly, the Synergistic
Polyphenol Compounds are useful for treating or preventing liver
cancer.
6.25 Example 25
Effect of EGCG and Polyphenone E on Ordered Membrane Domains in
HT29 Colon Cancer Cells
Pretreatment with DilC.sub.16
[0230] HT29 human colon cancer cells were treated with the
fluorescent lipid analog DilC.sub.16, which preferentially
incorporates into ordered domains in the plasma membrane.
Subsequent treatment of the cells with EGCG, an illustrative
Synergistic Polyphenol Compound, or Polyphenone E, an illustrative
Synergistic Polyphenol Compound, resulted in a marked increase in
the sensitivity of the plasma membrane to extraction using cold
Triton X-100, indicating that liquid ordered domains are
decreasing.
Pretreatment with a Synergistic Polyphenol Compound or
Composition
[0231] HT29 cells were pretreated with EGCG or Polyphenone E and
both compounds were shown to inhibit subsequent incorporation of
DilC.sub.16 into the plasma membrane, this result being indicative
of a change in membrane organization, induced by the EGCG or
Polyphenone E.
[0232] These effects were detected with as little as 2 mg/mL of
EGCG, and within 5 minutes. Phosphorylation of EGFR, ERK1, ERK2,
and AKT were suppressed in cells treated with
methyl-.beta.-cyclodextrin, which depletes plasma membrane
cholesterol. However, filipin staining indicated that the effects
of EGCG on plasma membrane properties are not associated with
cholesterol depletion. In addition, the inhibitory effect of EGCG
on phosphorylation of EGFR was not blocked by the addition of
cholesterol on the growth medium. These findings provide evidence
that EGCG can alter membrane domain organization in colon cancer
cells ant that this is not due to cholesterol depletion. It was
noted that pre-treatment of the cells with EGCG or Poly E inhibited
the binding of Alexa 488-labeled EGF to the cell surface receptor
EGFR. Accordingly, these data suggest that the Synergistic
Polyphenol Compounds and Compositions inhibit EGF binding, and
subsequent activation of the EGFR and downstream signaling pathway
by disrupting membrane domain organization.
[0233] The present invention is not to be limited in scope by the
specific embodiments disclosed in the examples, which are intended
as illustrations of a few aspects of the invention and any
embodiments that are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to one skilled in the art and are intended to fall within
the scope of the appended claims.
Sequence CWU 1
1
12 1 20 DNA Artificial Synthetic oligonucleotide 1 gttcccaccc
atgtcaaaac 20 2 20 DNA Artificial Synthetic oligonucleotide 2
gccctcgctt atgatctgtc 20 3 20 DNA Artificial Synthetic
oligonucleotide 3 gctacagcat gcagagcaag 20 4 20 DNA Artificial
Synthetic oligonucleotide 4 acgcccttgt ttcagaaatg 20 5 20 DNA
Artificial Synthetic oligonucleotide 5 gaaggtgaag atgcacacca 20 6
20 DNA Artificial Synthetic oligonucleotide 6 caggtaactc gtgcagagca
20 7 20 DNA Artificial Synthetic oligonucleotide 7 gagtgccaga
tgttgcagaa 20 8 20 DNA Artificial Synthetic oligonucleotide 8
tggggatctc catttccata 20 9 20 DNA Artificial Synthetic
oligonucleotide 9 ggcgctcatg taccctatgt 20 10 20 DNA Artificial
Synthetic oligonucleotide 10 tcaaagaccg agtccagctt 20 11 20 DNA
Artificial Synthetic oligonucleotide 11 tgctgttcat gaatggcttc 20 12
20 DNA Artificial Synthetic oligonucleotide 12 taagctcagg
accctgctgt 20
* * * * *